Preparation of polymer conjugates of therapeutic, agricultural, and food additive compounds

ABSTRACT

Disclosed is a process for preparing polymer conjugates of agricultural, therapeutic, and food additive compounds using Mitsunobu conditions.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to processes for making polymer conjugates oftherapeutic, agricultural, and food additive compounds. Morespecifically, the invention relates to processes employing Mitsunobureaction conditions to prepare conjugates for use in treating variousmammalian, particularly human, diseases and disorders as well as inagriculture and as food additives. In certain aspects, the inventionrelates to using Mitsunobu conditions with an alcohol, particularly analcohol-containing polymer, and an amine to form the desired conjugate.

2. State of the Art

Attachment of biologically active compounds to polymers has receivedsignificant attention and has become a common method to control variouscharacteristics, e.g., biodistribution, pharmacokinetics, and toxicity,of such compounds. A frequent choice of polymer for use in makingpolymer conjugates of biologically active compounds is polyethyleneglycol (PEG). It is widely used as a covalent modifier of both small andlarge biologically active molecules. For discussions of such conjugates,see, Eur. Polym. J. 19, No. 12, pages. 1177-1183 (Zaplinsky et al.,1983) et al., Journal of Controlled Release 10 (1989) 145-154 (Veroneseet al., 1989), and Advanced Drug Delivery Reviews, 16, 157-182(Zaplinsky, 1995).

It has recently been discovered that polymer conjugates of, for example,α₄β₁ (VLA-4) antagonists have greatly improved serum half-life. Thesepolymer compounds can be prepared using various synthetic methods,including carboxamide formation by reacting an ester of the activemolecule with a polymer amine, carbamate formation between an amine ofthe active molecule and a polymer chloroformate, or carbamate formationbetween an isocyanate of the active molecule and a polymer alcohol. Theoverall yields from these methods are typically less than desirable,often involving multiple steps and purification means. It is thereforenecessary to design a process which isolates a conjugate of a VLA-4inhibitor in quantitative or near quantitative yields.

The importance of such polymer conjugates indicates that there is a needfor convenient and efficient syntheses of such materials.

SUMMARY OF THE INVENTION

This invention provides an improved synthesis of polymer conjugates ofagricultural, therapeutic, and food additive compounds. The process ofthis invention produces the final conjugate products in excellentyields. In a preferred aspect, the invention provides a process for thepreparation of conjugates by condensing polymeric alcohols with aminesusing Mitsunobu reaction conditions. In another preferred aspect, theinvention provides a process for the preparation of conjugates bycondensing polymeric amines with alcohols using Mitsunobu reactionconditions.

In one aspect, the invention provides a process for the preparation ofconjugates of active compounds, comprising the steps of: a) reacting apolymeric alcohol with a nucleophile in the presence of atrivalentphosphine and an appropriate azodicarbonyl compound, e.g.,diethylazodicarboxylate or azodicarbonyldipiperidide, to form theconjugate; and b) isolating the conjugate.

In a specific aspect, the active compounds and the resulting conjugatesexhibit VLA-4 antagonistic properties.

In another aspect, the invention provides a process for the preparationof conjugates of formula I below:

B is a bio-compatible polymer moiety;q is from about 1 to about 100;A at each occurrence is independently a compound having biological oragricultural activity or A is a food additive compound.

This process comprises

-   -   a) reacting polymeric alcohol of formula Ia    -    with a nucleophile of formula H-Nu    -    in the presence of a trivalentphosphine and an appropriately        substituted azodicarbonyl compound to form the compound of        formula I, where Nu is a radical corresponding to formula A        above and the H is an acidic hydrogen on Nu; and    -   b) isolating the compound of formula I.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, this invention provides processes for the preparation ofconjugates of agricultural, therapeutic, and food additive compounds(hereinafter “active compounds”). The conjugates comprise one or morepolymer moieties covalently attached to one or more active compoundswhere the resulting conjugates have the same type of activity as theactive compound. In one aspect, the active compounds and resultingconjugates are compounds that inhibit leukocyte adhesion and, inparticular, leukocyte adhesion mediated, at least in part, by α₄integrins.

In a preferred aspect, the A group in the conjugates of Formula I can berepresented by formula II

wherein

-   J is selected from:    -   a) a group of formula (a):    -   wherein R³¹ is a covalent bond to the polymer moiety which        optionally comprises a linker, or R³¹ is —H, R^(31′), —NH₂,        —NHR^(31′) or —N(R^(31′))₂, —NC₃-C₆cyclic, —OR^(31′), —SR^(31′),        wherein each R^(31′) is independently an optionally substituted        straight or branched C₁-C₆alkyl, optionally substituted        C₃-C₆cycloalkyl, optionally substituted aryl, optionally        substituted heteroaryl,    -   and R³² is a covalent bond to the polymer moiety which        optionally comprises a linker, or R³² is —H, —NO₂, haloalkyl or        the group —N(MR⁴¹)R⁴² wherein M is a covalent bond, —C(O)— or        —SO₂—, R⁴¹ is R^(41′), N(R^(41′))₂, or —OR^(41′),    -   wherein each R^(41′) is independently hydrogen, an optionally        substituted straight or branched C₁-C₆alkyl, optionally        substituted cycloalkyl, optionally substituted aryl, optionally        substituted heterocyclic or an optionally substituted        heteroaryl, wherein optional substitutions are halide,        C₁-C₆alkyl, or —OC₁-C₆alkyl,    -   and R⁴² is hydrogen or R^(41′); and    -   b) a group of formula (b):    -   wherein R is selected from the group consisting of a covalent        bond to the polymer moiety, amino, substituted amino, alkyl and        substituted alkyl wherein each amino, substituted amino, alkyl        and substituted alkyl is optionally covalently bound to the        polymer moiety wherein, in each case, the polymer moiety        optionally comprises a linker which covalently links the polymer        moiety;    -   Ar¹ is selected from the group consisting of aryl, substituted        aryl, heteroaryl and substituted heteroaryl wherein each of        aryl, substituted aryl, heteroaryl and substituted heteroaryl is        optionally covalently bound to the polymer moiety wherein the        polymer moiety optionally comprises a linker which covalently        links the polymer moiety to Ar¹;-   Ar² is selected from the group consisting of aryl, substituted aryl,    heteroaryl and substituted heteroaryl wherein each of aryl,    substituted aryl, heteroaryl and substituted heteroaryl is    optionally covalently bound to the polymer moiety wherein the    polymer moiety optionally comprises a linker which covalently links    the polymer moiety to Ar²;    -   X is selected from the group consisting of —NR¹, —O—, —S—, —SO—,        —SO₂ and optionally substituted —CH₂— where R¹ is selected from        the group consisting of hydrogen and alkyl;-   T is selected from:    -   a) a group of formula (c)    -   wherein Y is selected from the group consisting of —O— and —NR¹—        wherein R¹ is selected from the group consisting of hydrogen and        alkyl;    -   W is selected from the group consisting of a covalent bond to a        polymer moiety which optionally comprises a linker and —NR²R³        wherein R² and R³ are independently selected from the group        consisting of hydrogen, alkyl, substituted alkyl, and where R²        and R³, together with the nitrogen atom bound thereto, form a        heterocyclic ring or a substituted heterocyclic ring wherein        each of alkyl, substituted alkyl, heterocyclic and substituted        heterocyclic is optionally covalently bound to a polymer moiety        which further optionally comprises a linker;    -   m is an integer equal to 0, 1 or 2;    -   n is an integer equal to 0, 1 or 2; and    -   b) a group of formula (d)    -   wherein G is an optionally substituted aryl or optionally        substituted heteroaryl 5 or 6 membered ring containing 0 to 3        nitrogens, wherein said aryl or heteroary optionally further        comprises a covalent bond to a polymer moiety which optionally        comprises a linker;    -   R⁶ is a covalent bond to a polymer moiety which optionally        comprises a linker, or R⁶ is —H;-   R⁵⁵ is selected from the group consisting of alkoxy, substituted    alkoxy, cycloalkoxy, substituted cycloalkoxy, aryloxy and    substituted aryloxy, and —OH;    provided that:

A. at least one of J, Ar¹, Ar², and T contains a covalent bond to thepolymer moiety;

B. when J is covalently bound to the polymer moiety, n is one and X isnot —O—, —S—, —SO—, or —SO₂—; and

C. when X is —O—, then m is two.

In a preferred embodiment, only one of J, Ar¹, Ar², and T contains acovalent bond to a polymer moiety.

The compound H-Nu employed in the conjugation reaction is a compoundcontaining an acidic hydrogen covalently bonded to Nu. Preferredcompounds of formula H-Nu can be represented by the formula II.1

whereinJ and Ar² carry the same definitions as set forth for Formula II;T is a group carrying an acidic hydrogen; andR⁵⁵ is a acid protecting group.

Suitable T groups include heterocyclic groups, imides, phenols,phosphoric mono- and diesters, carboxylic acids, hydroxamates, thiols,thioamides, β-keto esters, and 1,3-diketones,

A preferred T group is a group of formula (c)

-   -   wherein Y is selected from the group consisting of —O— and —NR¹—        wherein R¹ is selected from the group consisting of hydrogen and        alkyl;    -   W is a group containing an acidic hydrogen, preferably on a        nitrogen atom adjacent a carbonyl, where the W group optionally        connected to Y(CO) by a linker.

Another preferred T group is a group of formula (d)

-   -   wherein G is an optionally substituted aryl or optionally        substituted heteroaryl 5 or 6 membered ring containing 0 to 3        nitrogens; and    -   R⁶ is hydrogen.

It should be understood that the value for q is calculated based on theratio of the number of polymer moieties and the number of A-moieties. Inother words, when q is 1.5, this contemplates, for example, thefollowing formula I′:

Preferred conjugates of formula I prepared by the invention includethose of formula Ia below:

and pharmaceutically acceptable salts thereof, whereinB is a polymer moiety optionally covalently attached to a carrier;q is from about 1 to about 100;A at each occurrence is independently a compound of formula IIa

wherein

-   R is selected from the group consisting of a covalent bond to the    polymer moiety, amino, substituted amino, alkyl and substituted    alkyl wherein each amino, substituted amino, alkyl and substituted    alkyl is optionally covalently bound to the polymer moiety wherein,    in each case, the polymer moiety optionally comprises a linker which    covalently links the polymer moiety;-   Ar¹ is selected from the group consisting of aryl, substituted aryl,    heteroaryl and substituted heteroaryl wherein each of aryl,    substituted aryl, heteroaryl and substituted heteroaryl is    optionally covalently bound to the polymer moiety wherein the    polymer moiety optionally comprises a linker which covalently links    the polymer moiety to Ar¹;    Ar² is selected from the group consisting of aryl, substituted aryl,    heteroaryl and substituted heteroaryl wherein each of aryl,    substituted aryl, heteroaryl and substituted heteroaryl is    optionally covalently bound to the polymer moiety wherein the    polymer moiety optionally comprises a linker which covalently links    the polymer moiety to Ar²;-   X is selected from the group consisting of —NR¹—, —O—, —S—, —SO—,    —SO₂ and optionally substituted —CH₂— where R¹ is selected from the    group consisting of hydrogen and alkyl;-   Y is selected from the group consisting of —O— and —NR¹— wherein R¹    is selected from the group consisting of hydrogen and alkyl;-   W is selected from the group consisting of a covalent bond to a    polymer moiety which optionally comprises a linker and —NR²R³    wherein R² and R³ are independently selected from the group    consisting of hydrogen, alkyl, substituted alkyl, and where R² and    R³, together with the nitrogen atom bound thereto, form a    heterocyclic ring or a substituted heterocyclic ring wherein each of    alkyl, substituted alkyl, heterocyclic and substituted heterocyclic    is optionally covalently bound to a polymer moiety which further    optionally comprises a linker;-   m is an integer equal to 0, 1 or 2;-   n is an integer equal to 0, 1 or 2; and-   pharmaceutically acceptable salts thereof;    provided that:

A. at least one of R, Ar¹, Ar², W and —NR²R³ contain a covalent bond tothe polymer moiety;

B. when R is covalently bound to the polymer moiety, n is one and X isnot —O—, —S—, —SO—, or —SO₂—;

C. when X is —O— or —NR¹—, then m is two;. and

D. the conjugate of formula Ia has a molecular weight of no more than100,000.

Preferred conjugates of formula I prepared by the invention includethose of formula Ib below:

wherein each A is independently a compound of formula IIb below:

and wherein q is about 1 to about 100;

B is a polymer moiety optionally covalently attached to a carrier;

Ar¹ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the PEG moiety optionally comprises alinker which covalently links the PEG moiety to Ar¹;

Ar² is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the polymer moiety optionallycomprises a linker which covalently links the polymer moiety to Ar²;

Y is selected from the group consisting of —O— and —NR¹— wherein R¹ isselected from the group consisting of hydrogen and alkyl;

W is selected from the group consisting of a covalent bond to a polymermoiety which optionally comprises a linker and —NR²R³ wherein R² and R³are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, and where R² and R³, together with the nitrogen atombound thereto, form a heterocyclic ring or a substituted heterocyclicring wherein each of alkyl, substituted alkyl, heterocyclic andsubstituted heterocyclic is optionally covalently bound to the polymermoiety which further optionally comprises a linker;

provided that at least one of Ar¹, A², W and —NR²R³ is covalently boundto a polymer moiety which optionally comprises a linker;

and further provided that the conjugate of formula Ib has a molecularweight of no more than 100,000.

Preferred conjugates of formula I formed by the process of the inventioninclude those of formula Ic below:

wherein each A is independently a compound of formula IIc below:

and wherein q is about 1 to about 100;

B is a polymer moiety optionally covalently attached to a carrier;

R is selected from the group consisting of a covalent bond to a polymermoiety, amino, substituted amino, alkyl and substituted alkyl whereineach amino, substituted amino, alkyl and substituted alkyl is optionallycovalently bound to the polymer moiety wherein, in each case, thepolymer moiety optionally comprises a linker which covalently links thepolymer moiety;

Ar¹ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the polymer moiety optionallycomprises a linker which covalently links the polymer moiety to Ar¹;

Ar² is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the polymer moiety optionallycomprises a linker which covalently links the polymer moiety to Ar²;

Y is selected from the group consisting of —O— and —NR¹— wherein R¹ isselected from the group consisting of hydrogen and alkyl;

W is selected from the group consisting of a covalent bond to a polymermoiety which optionally comprises a linker and —NR²R³ where R² and R³,together with the nitrogen atom bound thereto, form a heterocyclic ringor a substituted heterocyclic ring wherein each of alkyl, substitutedalkyl, heterocyclic and substituted heterocyclic is optionallycovalently bound to a polymer moiety which further optionally comprisesa linker;

n is an integer equal to 0, 1 or 2; and

pharmaceutically acceptable salts thereof;

provided that at least one of R, Ar¹, Ar², W and —NR²R³ is covalentlybound to a polymer moiety which optionally comprises a linker;

and further provided that the conjugate of formula Ic has a molecularweight of no more than 100,000.

Preferred conjugates of formula I include those of formula Id below:

wherein each A is independently a compound of formula IId below:

and wherein q is about 1 to about 100;

B is a polymer moiety optionally covalently attached to a carrier;

R is selected from the group consisting of a covalent bond to a polymermoiety, amino, substituted amino, alkyl and substituted alkyl whereineach amino, substituted amino, alkyl and substituted alkyl is optionallycovalently bound to a polymer moiety wherein, in each case, the polymermoiety optionally comprises a linker which covalently links the polymermoiety;

Ar¹ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the polymer moiety optionallycomprises a linker which covalently links the polymer moiety to Ar¹;

Ar² is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the polymer moiety optionallycomprises a linker which covalently links the polymer moiety to Ar²;

R² and R³ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, and where R² and R³, together withthe nitrogen atom bound thereto, form a heterocyclic ring or asubstituted heterocyclic ring wherein each of alkyl, substituted alkyl,heterocyclic and substituted heterocyclic is optionally covalently boundto a polymer moiety which further optionally comprises a linker;

n is an integer equal to 0, 1 or 2; and

pharmaceutically acceptable salts thereof;

provided that at least one of R, Ar¹, Ar², and —NR²R³ is covalentlybound to a polymer which optionally comprises a linker;

and further provided that the conjugate of formula Id has a molecularweight of no more than 100,000.

Preferred conjugates of formula I include those of formula Ie below:

wherein each A is independently a compound of formula IIe below:

and wherein q is about 1 to about 100;

B is a polymer moiety optionally covalently attached to a carrier;

Ar¹ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the polymer moiety optionallycomprises a linker which covalently links the polymer moiety to Ar¹;

Ar² is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl wherein each of aryl, substitutedaryl, heteroaryl and substituted heteroaryl is optionally covalentlybound to a polymer moiety wherein the polymer moiety optionallycomprises a linker which covalently links the polymer moiety to Ar²;

R² and R³ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, and where R² and R³, together withthe nitrogen atom bound thereto, form a heterocyclic ring or asubstituted heterocyclic ring wherein each of alkyl, substituted alkyl,heterocyclic and substituted heterocyclic is optionally covalently boundto a polymer moiety which further optionally comprises a linker; and

pharmaceutically acceptable salts thereof;

provided that at least one of Ar¹, Ar² and —NR²R³ is covalently bound toa polymer moiety which optionally comprises a linker;

and further provided that the conjugate of formula Ie has a molecularweight of no more than 100,000.

Preferred conjugates of formula I include those of formula If below:

wherein each A is independently a compound of formula IIf below:

and wherein q is about 1 to about 100;

B is a polymer moiety optionally covalently attached to a carrier;

R⁴ is covalently bound to a polymer moiety which optionally comprises alinker;

R⁵ is selected from the group consisting of alkyl and substituted alkyl;

Ar³ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl;

X is selected from the group consisting of —NR¹—, —O—, —S—, —SO—, —SO₂and optionally substituted —CH₂— where R¹ is selected from the groupconsisting of hydrogen and alkyl;

m is an integer equal to 0, 1 or 2;

n is an integer equal to 0, 1 or 2; and

pharmaceutically acceptable salts thereof;

provided that:

A. when R is covalently bound to the polymer moiety, n is one and X isnot —O—, —S—, —SO—, or —SO₂—;

B. when X is —O— or —NR¹—, then m is two;. and

C. the conjugate of formula If has a molecular weight of no more than100,000.

Preferred conjugates of formula I include those of formula Ig below:

wherein each A is independently a compound of formula IIg below:

and wherein q is about 1 to about 100;

B is a polymer moiety optionally covalently attached to a carrier;

R⁴ is covalently bound to a polymer moiety which optionally comprises alinker;

R⁵ is selected from the group consisting of alkyl and substituted alkyl;

Ar³ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl;

n is an integer equal to 0, 1 or 2; and

pharmaceutically acceptable salts thereof;

provided that the conjugate of formula Ig has a molecular weight of nomore than 100,000.

Preferred conjugates of formula I include those of formula Ih below:

wherein each A is independently a compound of formula IIh below:

and wherein q is about 1 to about 100;

R⁴ is covalently bound to a polymer moiety which optionally comprises alinker;

Ar³ is selected from the group consisting of aryl, substituted aryl,heteroaryl and substituted heteroaryl;

pharmaceutically acceptable salts thereof;

provided that the conjugate of formula Ih has a molecular weight of nomore than 100,000.

Preferred conjugates of formula I include those of formula Ii below:

wherein each A is independently a compound of formula IIi below:

or a pharmaceutically acceptable salt thereof,

and provided that the conjugate of formula Ii has a molecular weight ofno more than 100,000.

Preferably, when Ar¹ is not bound to a polymer moiety, Ar¹ in formulaeIIa-IIe and Ar³ in formulae IIf-IIh is selected from the groupconsisting of:

-   phenyl,-   4-methylphenyl,-   4-t-butylphenyl,-   2,4,6-trimethylphenyl,-   2-fluorophenyl,-   3-fluorophenyl,-   4-fluorophenyl,-   2,4-difluorophenyl,-   3,4-difluorophenyl,-   3,5-difluorophenyl,-   2-chlorophenyl,-   3-chlorophenyl,-   4-chlorophenyl,-   3,4-dichlorophenyl,-   3,5-dichlorophenyl,-   3-chloro-4-fluorophenyl,-   4-bromophenyl,-   2-methoxyphenyl,-   3-methoxyphenyl,-   4-methoxyphenyl,-   3,4-dimethoxyphenyl,-   4-t-butoxyphenyl,-   4-(3′-dimethylamino-n-propoxy)-phenyl,-   2-carboxyphenyl,-   2-(methoxycarbonyl)phenyl,-   4-(H₂NC(O)—)phenyl,-   4-(H₂NC(S)—)phenyl,-   4-cyanophenyl,-   4-trifluoromethylphenyl,-   4-trifluoromethoxyphenyl,-   3,5-di-(trifluoromethyl)phenyl,-   4-nitrophenyl,-   4-aminophenyl,-   4-(CH₃C(O)NH—)phenyl,-   4-(phenylNHC(O)NH—)phenyl,-   4-amidinophenyl,-   4-methylamidinophenyl,-   4-[CH₃SC(═NH)—]phenyl,-   4-chloro-3-[H₂NS(O)₂—]phenyl,-   1-naphthyl,-   2-naphthyl,-   pyridin-2-yl,-   pyridin-3-yl,-   pyridin-4-yl,-   pyrimidin-2-yl,-   quinolin-8-yl,-   2-(trifluoroacetyl)-1,2,3,4-tetrahydroisoquinolin-7-yl,-   2-thienyl,-   5-chloro-2-thienyl,-   2,5-dichloro-4-thienyl,-   1-N-methylimidazol-4-yl,-   1-N-methylpyrazol-3-yl,-   1-N-methylpyrazol-4-yl,-   1-N-butylpyrazol-4-yl,-   1-N-methyl-3-methyl-5-chloropyrazol-4-yl,-   1-N-methyl-5-methyl-3-chloropyrazol-4-yl,-   2-thiazolyl and-   5-methyl-1,3,4-thiadiazol-2-yl.

Preferably, when A is of the formulae IIa, IIb, IIc, IId, and IIe, andAr¹ is bound to a polymer moiety, then Ar¹ is of the formula:—Ar¹-Z-(CH₂CHR⁷O)_(p)R⁸

wherein

Ar¹ is selected from the group consisting of aryl, substituted aryl,heteroaryl, and substituted heteroaryl,

Z is selected from the group consisting of a covalent bond, a linkinggroup of from 1 to 40 atoms, —O—, and —NR⁹—, where R⁹ is selected fromthe group consisting of hydrogen and alkyl,

R⁷ is selected from the group consisting of hydrogen and methyl;

R⁸ is selected from the group consisting of A, -(L)_(w)-polymer carrier,hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and—CH₂CHR⁷NR¹⁰R¹¹ where R⁷ is as defined above, R¹⁰ and R¹¹ areindependently selected from the group consisting of hydrogen and alkyl,A is represented by any of formulae IIa through IIh above, L is alinking group of from 1 to 40 atoms and w is zero or one: and

p is an integer of from about 100 to 2200, preferably from about200-1360.

When A is of the Formulae ha or IIf, and R is not bound to a polymermoiety, the substituent of the following formula:

where R⁵, X, m and n are as defined above, is preferably selected fromthe group consisting of azetidinyl, thiazolidinyl, piperidinyl,piperazinyl, morpholino, thiomorpholinyl, pyrrolidinyl,4-hydroxypyrrolidinyl, 4-oxopyrrolidinyl, 4-fluoropyrrolidinyl,4,4-difluoropyrrolidinyl, 4-(thiomorpholin-4-ylC(O)O—)pyrrolidinyl,4-[CH₃S(O)₂O—]pyrrolidinyl, 3-phenylpyrrolidinyl,3-thiophenylpyrrolidinyl, 4-amino-pyrrolidinyl, 3-methoxypyrrolidinyl,4,4-dimethylpyrrolidinyl, 4-N-Cbz-piperazinyl, 4-[CH₃S(O)₂-]piperazinyl,5,5-dimethylthiazolindin-4-yl, 1,1-dioxo-thiazolidinyl,1,1-dioxo-5,5-dimethylthiazolidin-2-yl and 1,1-dioxothiomorpholinyl.

Preferably, when A is of the formulae IIa and the substituent of theformula:

is bound to the PEG moiety, then preferably the substituent is of theformula:

wherein

m is an integer equal to zero, one or two;

Z is selected from the group consisting of a covalent bond, a linkinggroup of from 1 to 40 atoms, —O—, and —NR⁹—, where R⁹ is selected fromthe group consisting of hydrogen and alkyl,

R⁷ is selected from the group consisting of hydrogen and methyl;

R⁸ is selected from the group consisting of A, -(L)_(w)-polymer carrier,hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and—CH₂CHR⁷NR¹⁰R¹¹ where R⁷ is as defined above, R¹⁰ and R¹¹ areindependently selected from the group consisting of hydrogen and alkyl,A is represented by any of formulae IIa through IIh above, L is alinking group of from 1 to 40 atoms and w is zero or one: and

p is an integer of from about 100 to 2200, preferably from about200-1360.

When A is of the formula IIa, IIb, IIc, IId, IIe and when Ar² is notbound to a polymer moiety, then preferably Ar² is selected from thegroup consisting of phenyl, substituted phenyl, 2-pyridinyl,3-pyridinyl, 4-pyridinyl, and 4-pyridin-2-onyl.

When A is of the formula IIa, IIb, IIc, IId, IIe and when Ar² is boundto a polymer moiety, then Ar² is preferably represented by the formula:

where Ar² is selected from the group consisting of aryl, substitutedaryl, heteroaryl and substituted heteroaryl;

Z is selected from the group consisting of a covalent bond, a linkinggroup of from 1 to 40 atoms, —O—, and —NR⁹—, where R⁹ is selected fromthe group consisting of hydrogen and alkyl,

R⁷ is selected from the group consisting of hydrogen and methyl;

R⁸ is selected from the group consisting of A, -(L)_(w)-polymer carrier,hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and—CH₂CHR⁷NR¹⁰R¹¹ where R⁷ is as defined above, R¹⁰ and R¹¹ areindependently selected from the group consisting of hydrogen and alkyl,A is represented by any of formulae IIa through IIh above, L is alinking group of from 1 to 40 atoms and w is zero or one: and

p is an integer of from about 100 to 2200, preferably from about200-1360.

In one preferred embodiment of conjugates prepared by the inventiveprocess, —YC(O)W is —OC(O)NR²R³.

When A is of the formulae IIa, IIb, or IIc, —YC(O)W is —OC(O)NR²R³ andneither R² nor R³ are not bound to a polymer moiety, then preferably—OC(O)NR²R³ is selected from the group consisting of:

-   (CH₃)₂NC(O)O—,-   (piperidin-1-yl)-C(O)O—,-   (piperidin-4-yl)-C(O)O—,-   (1-methylpiperidin-4-yl)-C(O)O—,-   (4-hydroxypiperidin-1-yl)-C(O)O—,-   (4-formyloxypiperidin-1-yl)-C(O)O—,-   (4-ethoxycarbonylpiperidin-1-yl)-C(O)O—,-   (4-carboxylpiperidin-1-yl)-C(O)O—,-   (3-hydroxymethylpiperidin-1-yl)-C(O)O—,-   (4-hydroxymethylpiperidin-1-yl)-C(O)O—,-   (4-phenyl-1-Boc-piperidin-4-yl)-C(O)O—,-   (4-piperidon-1-yl ethylene ketal)-C(O)O—,-   (piperazin-4-yl)-C(O)O—,-   (1-Boc-piperazin-4-yl)-C(O)O—,-   (4-methylpiperazin-1-yl)-C(O)O—,-   (4-methylhomopiperazin-1-yl)-C(O)O—,-   (4-(2-hydroxyethyl)piperazin-1-yl)-C(O)O—,-   (4-phenylpiperazin-1-yl)-C(O)O—,-   (4-(pyridin-2-yl)piperazin-1)-yl)-C(O)O—,-   (4-(4-trifluoromethylpyridin-2-yl)piperazin-1-yl)-C(O)O—,-   (4-(pyrimidin-2-yl)piperazin-1-yl)-C(O)O—,-   (4-acetylpiperazin-1-yl)-C(O)O—,-   (4-(phenyl-C(O)-)piperazin-1-yl)-C(O)O—,-   (4-(pyridin-4′-yl-C(O)-)piperazin-1-yl)-C(O)O—,-   (4-(phenyl-NHC(O)-)piperazin-1-yl)-C(O)O—,-   (4-(phenyl-NHC(S)-)piperazin-1-yl)-C(O)O—,-   (4-methanesulfonylpiperazin-1-yl)-C(O)O—,-   (4-trifluoromethanesulfonylpiperazin-1-yl)-C(O)O—,-   (morpholin-4-yl)-C(O)O—,-   (thiomorpholin-4-yl)-C(O)O—, (thiomorpholin-4′-yl sulfone)-C(O)O—,-   (pyrrolidin-1-yl)-C(O)O—,-   (2-methylpyrrolidin-1-yl)-C(O)O—,-   (2-(methoxycarbonyl)pyrrolidin-1-yl)-C(O)O—,-   (2-(hydroxymethyl)pyrrolidin-1-yl)-C(O)O—,-   (2-(N,N-dimethylamino)ethyl)(CH₃)NC(O)O—,-   (2-(N-methyl-N-toluene-4-sulfonylamino)ethyl)(CH₃)N—C(O)O—,-   (2-(morpholin-4-yl)ethyl)(CH₃)NC(O)O—,-   (2-(hydroxy)ethyl)(CH₃)NC(O)O—,-   bis(2-(hydroxy)ethyl)NC(O)O—,-   (2-(formyloxy)ethyl)(CH₃)NC(O)O—,-   (CH₃OC(O)CH₂)HNC(O)O—, and-   2-(phenylNHC(O)O—)ethyl-]HNC(O)O—.

When A is of the formulae IIa, IIb, or IIc, —YC(O)W is —OC(O)NR²R³ andR² and/or R³ are/is bound to the PEG moiety, the PEG moiety ispreferably represented by the formula:-Z′-(CH₂CHR⁷O)_(p)R⁸

Z′ is selected from the group consisting of a covalent bond and alinking group of from 1 to 40 atoms;

R⁷ is selected from the group consisting of hydrogen and methyl;

R⁸ is selected from the group consisting of A, -(L)_(w)-polymer carrier,hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and—CH₂CHR⁷NR¹⁰R¹¹ where R⁷ is as defined above, R¹⁰ and R¹¹ areindependently selected from the group consisting of hydrogen and alkyl,A is represented by formula II above, L is a linking group of from 1 to40 atoms and w is zero or one: and

p is an integer of from about 100 to 2200.

Preferred T groups in Formula II.1 have formula (d) below.

where R⁶ is hydrogen.

Preferred (d) groups include those shown below in Table D. TABLE D

or

whereinR⁶⁶ is a covalent bond to a polymer moiety which optionally comprises alinker, orR⁶⁶ is hydrogen or straight or branched C₁-C₆alkyl;R⁷⁷ is a covalent bond to a polymer moiety which optionally comprises alinker, orR⁷⁷ is hydrogen, halogen or straight or branched C₁-C₆alkoxy; andR⁸⁸ is hydrogen.

Other (d) groups useful in the invention include those shown in TableD1. TABLE D1

Other preferred compounds of formula II.1 for use in the processes ofthe invention have formula II.1-a:

II.1-a

and pharmaceutically acceptable salts thereof, wherein

-   R⁵⁵ is an acid protecting group, preferably C₁-C₆ alkoxy, more    preferably C₂-C₄ alkoxy;-   Ar¹ is selected from the group consisting of alkyl, substituted    alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,    heterocyclic, substituted heterocyclic, heteroaryl and substituted    heteroaryl; and-   R⁶ is hydrogen.

Preferred compounds of Formula II.1-a include those wherein Ar¹ isphenyl or a 5- or 6-membered heteroaryl group having at least onenitrogen atom, each of which is optionally substituted with halogen,hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkyl, nitro, trifluoromethyl, amino, mono-or di(C₁-C₆)alkylamino, amino(C₁-C₆)alkyl, C₂-C₆ acyl, C₂-C₆ acylamino,or amino(C₁-C₆)acyl. Ar¹ is pyridyl optionally substituted with halogen,hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, nitro, trifluoromethyl, amino, mono-or di(C₁-C₆)alkylamino, amino(C₁-C₆)alkyl, C₂-C₆ acyl, C₂-C₆ acylamino,or amino(C₁-C₆)acyl. Particularly preferred compounds of Formula II.1-ainclude those where Ar¹ is pyridyl optionally substituted with C₁-C₆alkyl, hydroxy, halogen, C₁-C₆ alkoxy, nitro, trifluoromethyl, amino, ormono- or di(C₁-C₆)alkylamino.

Still other preferred compounds of formula II.1 are also those of theformula II.2-a:

and pharmaceutically acceptable salts thereof, whereinR⁵⁵ is an acid protecting group, preferably C₁-C₆ alkoxy, morepreferably C₂-C₄ alkoxy;R⁶ is hydrogen.

Preferred compounds of Formula II.2-a include those where R³¹ is aminoor mono- or di(C₁-C₆)alkylamino; and R³² is —H, —NO₂ or haloalkyl, morepreferably trifluoromethylmethyl.

Still other preferred compounds of Formula II.2-a are those where

-   R³¹ is amino or mono- or di(C₁-C₆)alkylamino; and-   R³² is —N(MR⁴¹)R⁴²; where M is —SO₂— or —CO—;-   R⁴¹ is C₁-C₆ alkyl optionally substituted with halogen, hydroxy,    C₁-C₆ alkoxy, amino, or mono- or di(C₁-C₆)alkylamino; or    -   phenyl or a 5- or 6-membered heteroaryl containing at least one        nitrogen, each of which is optionally substituted with halogen,        hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₇ cycloalkyl, amino,        nitro, trifluoromethyl, or mono- or di(C₁-C₆)alkylamino; and-   R⁴² is hydrogen, C₁-C₆alkyl, or C₃-C₇cycloalkyl. Further preferred    compounds of formula II.2-a include those wherein-   R⁴¹ groups within Formula II.2-a are C₁-C₄ alkyl optionally    substituted with halogen, hydroxy, C₁-C₆ alkoxy, amino, or mono- or    di(C₁-C₆)alkylamino; or    -   pyridyl or pyrimidinyl, each of which is optionally substituted        with halogen, hydroxy, C₁-C₃ alkyl, C₁-C₃ alkoxy, amino, or        mono- or di(C₁-C₄)alkylamino; and-   R⁴² is hydrogen, C₁-C₄alkyl, or C₃-C₇cycloalkyl.

In one example, the conjugates of this invention are divalent and arerepresented by formula III:

where each A is independently as defined above and B′ is-Z′-(CH₂CHR⁷O)_(p)-Z′- where each Z′ is independently a covalent bond ora linking group, R⁷ is hydrogen or methyl and p is an integer of fromabout 100 to 1360.

In another example, the conjugates of this invention are trivalent todecavalent and are preferably represented by formula IV:

where each A is independently as defined above and t is an integer from3 to 10.

The process of the instant invention employs the Mitsunobu reaction, acondensation reaction of alcohols in the presence of a triaryl- ortrialkylphosphine and an appropriate azodicarboxylate. In preferredreactions, polymer alcohols, e.g, polyethylene glycol, are reacted withnucleophiles in the presence of a triaryl- or trialkylphosphine and thediazo reagent to afford the conjugates. Nucleophiles are compoundshaving an acidic hydrogen, i.e., a moiety suitable to donate electrons.The bond formed by reacting a polymeric alcohol with a nucleophile canbe diverse, such as, for example, carbon-oxygen bond formation,carbon-nitrogen bond formation, carbon-sulfur bond formation,carbon-halogen bond formation, and carbon-carbon bond formation.

Thus, non-derivatized active compounds can have a wide variety offunctional groups that can react with the polymer alcohols. Examplesinclude carboxylic acids, alcohols, β-ketoesters, amines, thiols, alkylhalides, acyl halides, -diketones and the like. Other examples ofnucleophiles that can be utilized in the process of the presentinvention can be found in Organic Reactions, 1992, 42, 335-656, which isincorporated herein by reference in its entirety.

Examples of triaryl- or trialkylphosphines are described in Synthesis,2003, 3, 317-334; Tetrahedron Lett., 1998, 39, 7787; Chem. Commun. 1997,759; and Nucleosides Nucleotides, 1999, 18, 727, all incorporated hereinby reference and include triphenylphosphine, trimethylphosphine,triethylphosphine, tributylphosphine, and1,2-bis-(diphenylphosphino)ethane. The phosphines can also be polymersupported or water soluble. A preferred triarylphosphine istriphenylphosphine.

The diazo reagents are generally esters or amides of azodicarboxylicacids, and include those found in Synthesis, 2003, 3, 317-334;Tetrahedron Lett., 1999, 40, 7359; and Bull. Chem. Soc. Jpn., 1984, 57,2675. Specific examples of such diazo compounds arediethyldiazocarboxylate, diisopropylazodicarboxylate,4-methyl-1,2,4-triazolidine-3,5-dione,N,N,N′,N′-tetramethylazodicarboxamide, azodicarboxylic aciddipiperidide, bis(N-4-methylpiperazin-1-yl)azodicarboxamide,dimorpholinoazodicarboxamide and di-tert-butyl azodicarboxylate.

Representative conjugates prepared by the process of the instantinvention, including pharmaceutically acceptable salts thereof, are setforth in the following table: TABLE I

Comp. B A A

B

C

D

E

where in each of the structures the sum of all p's is from 100 to 2200,preferably from about 200-1360.

Definitions

As used herein, “alkyl” refers to monovalent saturated aliphatichydrocarbyl groups having from 1 to 5 carbon atoms and more preferably 1to 3 carbon atoms. This term is exemplified by groups such as methyl,ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl and the like.

“Substituted alkyl” refers to an alkyl group having from 1 to 3, andpreferably 1 to 2, substituents selected from the group consisting ofalkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy,cyano, halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl,substituted cycloalkyl, spirocycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupspreferably having from 1 to 5 and more preferably 1 to 3 carbon atomswhich are either straight-chained or branched. This term is exemplifiedby groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—) and the like.

“Alkoxy” refers to the group “alkyl-O-” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy,sec-butoxy, n-pentoxy and the like.

“Substituted alkoxy” refers to the group “substituted alkyl-O-”.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)— cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O)—, heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Aminoacyl” refers to the group —C(O)NR¹⁰R¹⁰ where each R¹⁰ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic and where each R10 is joined to form together with thenitrogen atom a heterocyclic or substituted heterocyclic ring whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substitutedheteroaryl-C(O)O—, heterocyclic-C(O)O—, and substitutedheterocyclic-C(O)O-wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Alkenyl” refers to alkenyl groups having from 2 to 6 carbon atoms andpreferably 2 to 4 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkenyl unsaturation. Such groups are exemplified byvinyl, allyl, but-3-en-1-yl, and the like.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic withthe proviso that any hydroxyl substitution is not attached to a vinyl(unsaturated) carbon atom.

“Alkynyl” refers to alkynyl groups having from 2 to 6 carbon atoms andpreferably 2 to 3 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkynyl unsaturation.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic.

“Amino” refers to the group —NH₂.

“Cyano” refers to the group —CN.

“Substituted amino” refers to the group —NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic and where R′ and R″ are joined, together with the nitrogenbound thereto to form a heterocyclic or substituted heterocyclic groupprovided that R′ and R″ are both not hydrogen. When R′ is hydrogen andR″ is alkyl, the substituted amino group is sometimes referred to hereinas alkylamino. When R′ and R″ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R′ or R″ is hydrogen butnot both. When referring to a disubstituted amino, it is meant thatneither R′ or R″ is hydrogen.

“Aminoacyl” refers to the groups —NR¹¹C(O)alkyl, —NR¹¹C(O)substitutedalkyl, —NR¹¹C(O)cycloalkyl, —NR¹¹C(O)substituted cycloalkyl,—NR¹¹C(O)alkenyl, —NR¹¹C(O)substituted alkenyl, —NR¹¹C(O)alkynyl,—NR¹¹C(O)substituted alkynyl, —NR¹¹C(O)aryl, —NR¹¹C(O)substituted aryl,—NR¹¹C(O)heteroaryl, —NR¹¹C(O)substituted heteroaryl,—NR¹¹C(O)heterocyclic, and —NR¹¹C(O)substituted heterocyclic where R¹¹is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Nitro” refers to the group —NO₂.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryls includephenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with from1 to 3 substituents, and preferably 1 to 2 substituents, selected fromthe group consisting of hydroxy, acyl, acylamino, acyloxy, alkyl,substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, amino, substituted amino,aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy,carboxyl, carboxyl esters, cyano, thiol, thioalkyl, substitutedthioalkyl, thioaryl, substituted thioaryl, thioheteroaryl, substitutedthioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl,thioheterocyclic, substituted thioheterocyclic, cycloalkyl, substitutedcycloalkyl, halo, nitro, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy, aminosulfonyl (NH₂—SO₂—), and substituted amino sulfonyl.

“Aryloxy” refers to the group aryl-O— that includes, by way of example,phenoxy, naphthoxy, and the like.

“Substituted aryloxy” refers to substituted aryl-O— groups.

“Carboxyl” refers to —COOH or salts thereof.

“Carboxyl ester” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)-aryl, and —C(O)O-substituted aryl wherein alkyl,substituted alkyl, aryl and substituted aryl are as defined herein.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including, by way of example,adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and thelike.

“Cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 10 carbonatoms having single or multiple cyclic rings and further having at least1 and preferably from 1 to 2 internal sites of ethylenic or vinyl(>C═C<) unsaturation.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refers to ancycloalkyl or cycloalkenyl group, having from 1 to 5 substituentsselected from the group consisting of oxo (═O), thioxo (═S), alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano,halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl,substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic, and substituted heterocyclic.

“Cycloalkoxy” refers to —O-cycloalkyl groups.

“Substituted cycloalkoxy” refers to —O-substituted cycloalkyl groups.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is fluoro or chloro.

“Hydroxy” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group.Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl,and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 3 substituents selected from the same groupof substituents defined for substituted aryl.

“Heteroaryloxy” refers to the group —O-heteroaryl and “substitutedheteroaryloxy” refers to the group —O-substituted heteroaryl.

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or unsaturated group having a single ring ormultiple condensed rings, from 1 to 10 carbon atoms and from 1 to 4hetero atoms selected from the group consisting of nitrogen, sulfur oroxygen within the ring wherein, in fused ring systems, one or more therings can be cycloalkyl, aryl or heteroaryl provided that the point ofattachment is through the heterocyclic ring.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 3 of the same substituents as defined forsubstituted cycloalkyl.

Examples of heterocyclyls and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydro-isoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and thelike.

“Thiol” refers to the group —SH.

“Thioalkyl” or “alkylthioether” or “thioalkoxy” refers to the group—S-alkyl.

“Substituted thioalkyl” or “substituted alkylthioether” or “substitutedthioalkoxy” refers to the group —S-substituted alkyl.

“Thioaryl” refers to the group —S-aryl, where aryl is defined above.

“Substituted thioaryl” refers to the group —S-substituted aryl, wheresubstituted aryl is defined above.

“Thioheteroaryl” refers to the group —S-heteroaryl, where heteroaryl isas defined above.

“Substituted thioheteroaryl” refers to the group —S-substitutedheteroaryl, where substituted thioheteroaryl is defined above.

“Thioheterocyclic” refers to the group —S-heterocyclic and “substitutedthioheterocyclic” refers to the group —S-substituted heterocyclic, whereheterocyclic and substituted heterocyclic.

“Heterocyclyloxy” refers to the group heterocyclyl-O— and “substitutedheterocyclyl-O-refers to the group substituted heterocyclyl-O— whereheterocyclyl and substituted heterocyclyl are as defined above.

“Thiocycloalkyl” refers to the group —S-cycloalkyl and “substitutedthiocycloalkyl” refers to the group —S-substituted cycloalkyl, wherecycloalkyl and substituted cycloalkyl are as defined above.

The terms “compound” and “active compound” are used to refer to theVLA-4 antagonist portion of a conjugate of the invention or to a VLA-4antagonist as it exists prior to conjugation to a polymer.

The terms “Linker”, “linking group” or “linker of from 1 to 40 atoms”refer to a group or groups that (1) covalently links the polymer to theactive compound and/or (2) covalently link the polyalkylene oxidemoieties of a polymer one to another. Within any particular conjugate,the linker connecting the polyalkylene oxide moieties of a polymertogether, and the linker bonding a polymer to an active compound may bethe same or different (i.e., may have the same or different chemicalstructures). Representative functional group linkages, of which alinking group may have one or more, are amides, ethers, carbamates,thiocarbamates, ureas, thioureas, amino groups, carbonyl groups, alkoxygroups, etc. The linker may be homogenous or heterogeneous in its atomcontent (e.g., linkers containing only carbon atoms or linkerscontaining carbon atoms as well as one or more heteroatoms present onthe linker. Preferably, the linker contains 1 to 25 carbon atoms and 0to 15 heteroatoms selected from oxygen, NR²², sulfur, —S(O)— and—S(O)₂—, where R²² is as defined above. The linker may also be chiral orachiral, linear, branched or cyclic.

Intervening between the functional group linkages or bonds within thelinker, the linker may further contain spacer groups including, but notlimited to, spacers selected from alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic, andcombinations thereof. The spacer may be homogenous or heterogeneous inits atom content (e.g., spacers containing only carbon atoms or spacerscontaining carbon atoms as well as one or more heteroatoms present onthe spacer. Preferably, the spacer contains 1 to 25 carbon atoms and 0to 15 heteroatoms selected from oxygen, NR²², sulfur, —S(O)— and—S(O)₂—, where R²² is as defined above. The spacer may also be chiral orachiral, linear, branched or cyclic.

Non-limiting examples of spacers are straight or branched alkylenechains, phenylene, biphenylene, etc. rings, all of which are capable ofcarrying one or more than one functional group capable of forming alinkage with the active compound and one or more polyalkylene oxidemoieties. One particular example of a polyfunctional linker-spacer groupis lysine, which may link any of the active compounds to two polymermoieties via the two amino groups substituted on a C₄ alkylene chain.Other non-limiting examples include p-aminobenzoic acid and3,5-diaminobenzoic acid which have 2 and 3 functional groupsrespectively available for linkage formation. Other such polyfunctionallinkage plus spacer groups can be readily envisaged by one of skill inthe art.

The term “polymer” refers to biocompatible, water-soluble, substantiallynon-immunogenic, polymers which are capable of being coupled to morethan one VLA-4 antagonists of formula II. Preferably the polymer isnon-ionic and biocompatible as measured by lack of toxicity at the dosesused. Inclusive of such polymers are multiple copies of polymers coupledto a carrier.

Examples of suitable polymers include, but are not limited to:polyoxyalkylene polymers such as polyethylene glycol (PEG),polyvinylpyrrolidone (PVP), polyacrylamide (PAAm),polydimethylacrylamide (PDAAm), polyvinyl alcohol (PVA), dextran, poly(L-glutamic acid) (PGA), styrene maleic anhydride (SMA),poly-N-(2-hydroxypropyl)methacrylamide (HPMA), polydivinylether maleicanhydride (DIVEMA) (Kameda, Y. et al., Biomaterials 25: 3259-3266, 2004;Thanou, M. et al, Current Opinion in Investigational Drugs 4(6):701-709, 2003; Veronese, F. M., et al., II Farmaco 54: 497-516, 1999).

Preferred polymers are polyoxyalkylenes. By “polyoxyalkylenes” is meantmacromolecules that include at least one polyalkylene oxide portion thatis optionally covalently bonded to one or more additional polyakyleneoxides, wherein the polyalkylene oxides are the same or different.Non-limiting examples include polyethylene glycol (PEG), polypropyleneglycol (PPG), polyisopropylene glycol (PIPG), PEG-PEG, PEG-PPG,PPG-PIPG, and the like. Also included within the definition ofpolyoxyalkylenes are macromolecules wherein the polyalkylene oxideportions are optionally connected to each other by a linker.Illustrative examples are PEG-linker-PEG, PEG-linker-PIPG, and the like.More specific examples include the commercially availablepoly[di(ethylene glycol)adipates, poly[di(ethylene glycol)phthalatediols, and the like. Other examples are block copolymers of oxyalkylene,polyethylene glycol, polypropylene glycol, and polyoxyethylenated polyolunits.

At at least one of its termini, the polymer is covalently attached tonon-polymer substituted compound of Formula II optionally through alinker using the process of the instant invention providing for covalentlinkage of the polymer to the non-polymer substituted compound ofFormula II.

When a linker is employed, the linker is covalently bonded to at leastone of the polymer termini which, in turn, is covalently attached to theotherwise, non-polymer substituted compound of Formula II. It isunderstood, of course, that if the appropriate substituents are found onthe non-polymer substituted compound of Formula II then the optionallinker may not be needed as there can be direct linkage of the polymerto the non-polymer substituted compound of Formula II.

Preferred linkers include, by way of example, the following —O—, —NR²²—,—NR²²C(O)O—, —OC(O)NR²²—, —NR²²C(O)—, —C(O)NR²²—, —NR²²C(O)NR²²—,-alkylene-NR²²C(O)O—, -alkylene-NR²²C(O)NR²²—, -alkylene-OC(O)NR²²—,-alkylene-NR²²—, -alkylene-O—, -alkylene-NR²²C(O)—, -alkylene-C(O)NR²²—,—NR²²C(O)O-alkylene-, —NR²²C(O)NR²²-alkylene-, —OC(O)NR²²-alkylene,—NR²²-alkylene-, —O-alkylene-, —NR²²C(O)-alkylene-, —C(O)NR²²-alkylene-,-alkylene-NR²²C(O)O-alkylene-, -alkylene-NR²²C(O)NR²²-alkylene-,-alkylene-OC(O)NR²²-alkylene-, -alkylene-NR²²-alkylene-,alkylene-O-alkylene-, -alkylene-NR^(22C)(O)-alkylene-,—C(O)NR²²-alkylene-, and

where

is selected from the group consisting of aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic, and D and E are independentlyselected from the group consisting of a bond, —O—, CO, —NR²²—,—NR²²C(O)O—, —OC(O)NR²²—, —NR²²C(O)—, —C(O)NR²²—, —NR²²C(O)NR²²—,-alkylene-NR²²C(O)O—, -alkylene-NR²²C(O)NR²²—, -alkylene-OC(O)NR²²—,-alkylene-NR²²—, -alkylene-O—, -alkylene-NR²²C(O)—, alkylene-C(O)NR²²—,—NR²²C(O)O-alkylene-, —NR²²C(O)NR²²-alkylene-, —OC(O) NR²²-alkylene-,—NR²²-alkylene-, —O-alkylene-, —NR²²C(O)-alkylene-, —C(O)NR²²-alkylene-,-alkylene-NR²²C(O)O-alkylene-, -alkylene-NR²²C(O)NR²²-alkylene-,-alkylene-OC(O)NR²²-alkylene-, -alkylene-NR²²-alkylene-,alkylene-O-alkylene-, -alkylene-NR²²C(O)-alkylene-, and—C(O)NR²²-alkylene-, where R²² is as defined above.

Preferred alkylene groups in the above linkers include C₁-C₁₅ alkylenegroups, more preferably C₁-C₆ alkylene groups, and most preferably C₁-C₃alkylene groups. Preferred heterocyclic groups include piperazinyl,piperidinyl, homopiperazinyl, homopiperidinyl, pyrrolidinyl, andimidazolidinyl.

The conjugates of the invention may be incorporated into a carrier whichmay have from 1 to 19 additional conjugates bound thereto. The term“carrier” refers to an optional component or scaffold to which multipleconjugates can be bound and which when incorporated into the conjugatesof this invention do not impart either substantial immunogenicity ortoxicity. Such carriers are preferably mono- to decavalent materialscontaining multiple functionalities for polymer attachment. Thefunctionalities can be homogeneous or heterogeneous; althoughhomogeneous functionalities are preferred.

Examples of commercially available carriers comprising homogeneousfunctionalities include, by way of example only, catechol, resorcinol,1,2-phenylenediamine, 1,3-phenylenediamine, 1,4-phenylenediamine;phthalic acid, isophthalic acid, 1,3-propanediol, glycerol,1,2,4-benzenetriol, pentaerythritol, glucose (in its pyranose form),1,3-phenylene diisocyanate, 1,4-phenylene diisocyanate,isophthalaldehyde, phthalaldehyde, 1,3-cyclopentanediol, ethylenediamine, ethylenediamine tetraacetic acid, and the like.

Representative structures useful as carriers or scaffolds include thefollowing (where PEG is used for representative purposes only):

Examples of commercially available carriers comprising heterogeneousfunctionalities include, by the way of example only, 6-hydroxycaproicacid, amino acids, salicylic acid, 3- or 4-aminosalicylic acid,1,3-diamino-2-hydroxypropane, 2-aminoethanol, 3-aminopropanol,glucosamine, sialic acid, amino acids, and the like.

Representative structures arising from polymer attachment using suchcarriers or scaffolds include the following (where PEG is used forrepresentative purposes only):

The carrier can optionally contain one or more copies of A bound to thecarrier optionally through a linker provided that there is at least onefurther functional group which can bind to a further copy of A. Forexample, each of the following structures is deemed a carrier orscaffold because there is at least one additional functional group forbinding of an additional A substituent:

where L, w and A are as defined above.

The term “oxyalkylene” refers to —OCH₂CHR_(d)- where R^(d) is alkyl.Polymerized oxyalkylenes are referred to as polyoxyalkylenes,polyalkylene oxides or polyalkylene glycols, non-limiting examples ofwhich include PEG, poly propylene glycol, polybutylene glycol,polyisopropylene glycol, and the like.

Such polymers are optionally mono-capped with a substituent preferablyselected from alkyl, aryl, substituted alkyl, substituted aryl and acarrier as described above. Inclusive of such polymers are those diaminocapped polyoxyalkylene polymers which are known in the art asJeffamines®. Still further, such polymers can optionally contain one ormore non-oxyalkylene units such as the commercially availablepoly[di(ethylene glycol)adipates, poly[di(ethylene glycol)phthalatediols, and the like. Also included are block copolymers of oxyalkylene,polyethylene glycol, polypropylene glycol, and polyoxyethylenated polyolunits.

Polyoxyalkylenes, such as PEG, are usually provided as a water soluble,waxy solid. Generally, as the polymer's molecular weight increases, itsviscosity and freezing point also increase. Commercial preparations areusually characterized by the “average molecular weight” of theconstituent polymers.

Typically, the average molecular weight of the total amount of polymerarising from single or multiple polymer moieties in the conjugates ofthe invention is between about 100 to 100,000; preferably from about10,000 to 80,000; more preferably from about 20,000 to about 70,000.

Similarly, other suitable polymers such polyvinylpyrrolidone (PVP),polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), polyvinyl alcohol(PVA), dextran, poly (L-glutamic acid) (PGA), styrene maleic anhydride(SMA), poly-N-(2-hydroxypropyl)methacrylamide (HPMA), polydivinylethermaleic anhydride (DIVEMA) are well known in the art and have molecularweights of from about 100 to 100,000; preferably from about 10,000 to80,000; more preferably from about 20,000 to about 70,000.

“Pharmaceutically acceptable salt” refers to salts which retain thebiological effectiveness and properties of the compounds of thisinvention and which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl)amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl)amines,cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines,di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substitutedcycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstitutedcycloalkenyl amines, aryl amines, diaryl amines, triaryl amines,heteroaryl amines, diheteroaryl amines, triheteroaryl amines,heterocyclic amines, diheterocyclic amines, triheterocyclic amines,mixed di- and tri-amines where at least two of the substituents on theamine are different and are selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl,heterocyclic, and the like. Also included are amines where the two orthree substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group.

Examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine,tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike. It should also be understood that other carboxylic acidderivatives would be useful in the practice of this invention, forexample, carboxylic acid amides, including carboxamides, lower alkylcarboxamides, dialkyl carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

The term “pharmaceutically-acceptable cation” refers to the cation of apharmaceutically-acceptable salt.

It is understood that in all substituted groups defined herein, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to -substituted aryl-(substitutedaryl)-(substituted aryl).

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups or a hydroxyl group alpha to ethenylic oracetylenic unsaturation). Such impermissible substitution patterns arewell known to the skilled artisan.

Compound Preparation

The starting active compounds employed in the process of this inventioncan be prepared from readily available starting materials using knownprocedures and readily available starting materials or, where thestarting material is not known or commercially available, such materialscan readily be prepared using literature procedures. It will beappreciated that where typical or preferred process conditions (i.e.,reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be usedunless otherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

Furthermore, the compounds of this invention will typically contain oneor more chiral centers. Accordingly, if desired, such compounds can beprepared or isolated as pure stereoisomers, i.e., as individualenantiomers or diastereomers, or as stereoisomer-enriched mixtures. Allsuch stereoisomers (and enriched mixtures) are included within the scopeof this invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

Preferred conjugates prepared according to this invention comprise apolymer moiety/optional carrier containing about 1 to about 100substituents of Formula II:

Specifically, the polymer moiety can be bound through a covalent bond tothe Ar¹ substituent, the R substituent, the Ar² substituent and/or inthe T substituent wherein the polymer moiety is either directly attachedor is attached via a linker. In turn, the polymer moiety may optionallybe bound to a carrier having multiple copies of the polymer attachedthereto.

Compounds of Formula II can be prepared by first coupling a heterocyclicamino acid, 1, with an appropriate aryl sulfonyl chloride as illustratedin Scheme 1 below:

where R, Ar¹, X, m and n are as defined above.

Specifically, in Scheme 1 above, heterocyclic amino acid, 1, is combinedwith a stoichiometric equivalent or excess amount (preferably from about1.1 to about 2 equivalents) of arylsulfonyl halide, 2, in a suitableinert diluent such as dichloromethane and the like. Generally, thereaction is conducted at a temperature ranging from about −70° C. toabout 40° C. until the reaction is substantially complete, whichtypically occurs within 1 to 24 hours. Preferably, the reaction isconducted in the presence of a suitable base to scavenge the acidgenerated during the reaction. Suitable bases include, by way ofexample, tertiary amines, such as triethylamine, diisopropylethylamine,N-methyl-morpholine and the like. Alternatively, the reaction can beconducted under Schotten-Baumann-type conditions using an aqueous alkalisolution such as an aqueous solution of sodium hydroxide, an aqueousphosphate solution buffered to pH 7.4, and the like. The resultingproduct, 3, can be recovered by conventional methods, such aschromatography, filtration, evaporation, crystallization, and the likeor, alternatively, used in the next step without purification and/orisolation.

Heterocyclic amino acids, 1, employed in the above reaction are eitherknown compounds or compounds that can be prepared from known compoundsby conventional synthetic procedures. Examples of suitable amino acidsfor use in this reaction include, but are not limited to, L-proline,trans-4-hydroxyl-L-proline, cis-4-hydroxyl-L-proline,trans-3-phenyl-L-proline, cis-3-phenyl-L-proline, L-(2-methyl)proline,L-pipecolinic acid, L-azetidine-2-carboxylic acid,L-thiazolidine-4-carboxylic acid,L-(5,5-dimethyl)thiazolidine-4-carboxylic acid,L-thiamorpholine-3-carboxylic acid. If desired, the correspondingcarboxylic acid esters of the amino acids, 1, such as the methyl esters,ethyl esters, t-butyl esters, and the like, can be employed in the abovereaction with the arylsulfonyl chloride. Subsequent hydrolysis of theester group to the carboxylic acid using conventional reagents andconditions, i.e., treatment with an alkali metal hydroxide in an inertdiluent such as methanol/water, then provides the N-sulfonyl amino acid,3.

Similarly, the arylsulfonyl chlorides, 2, employed in the above reactionare either known compounds or compounds that can be prepared from knowncompounds by conventional synthetic procedures. Such compounds aretypically prepared from the corresponding sulfonic acid, i.e., fromcompounds of the formula Ar¹SO₃H where Ar¹ is as defined above, usingphosphorous trichloride and phosphorous pentachloride. This reaction isgenerally conducted by contacting the sulfonic acid with about 2 to 5molar equivalents of phosphorous trichloride and phosphorouspentachloride, either neat or in an inert solvent, such asdichloromethane, at temperature in the range of about 0° C. to about 80°C. for about 1 to about 48 hours to afford the sulfonyl chloride.Alternatively, the arylsulfonyl chlorides, 2, can be prepared from thecorresponding thiol compound, i.e., from compounds of the Ar¹—SH whereAr¹ is as defined herein, by treating the thiol with chlorine (Cl₂) andwater under conventional reaction conditions.

Alternatively, arylsulfonyl chlorides, 2, employed in the above reactionmay be prepared by chlorosulfonylation of substituted benzene orheterocycloalkyl group using C₁—SO₃H.

Examples of arylsulfonyl chlorides suitable for use in this inventioninclude, but are not limited to, benzenesulfonyl chloride,1-naphthalenesulfonyl chloride, 2-naphthalenesulfonyl chloride,p-toluenesulfonyl chloride, o-toluenesulfonyl chloride,4-acetamidobenzenesulfonyl chloride, 4-tert-butylbenzenesulfonylchloride, 4-bromobenzenesulfonyl chloride, 2-carboxybenzenesulfonylchloride, 4-cyanobenzenesulfonyl chloride, 3,4-dichlorobenzenesulfonylchloride, 3,5-dichlorobenzenesulfonyl chloride,3,4-dimethoxybenzenesulfonyl chloride,3,5-ditrifluoromethylbenzenesulfonyl chloride, 4-fluorobenzenesulfonylchloride, 4-methoxybenzenesulfonyl chloride,2-methoxycarbonylbenzenesulfonyl chloride, 4-methylamido-benzenesulfonylchloride, 4-nitrobenzenesulfonyl chloride,4-trifluoromethyl-benzenesulfonyl chloride,4-trifluoromethoxybenzenesulfonyl chloride,2,4,6-trimethylbenzenesulfonyl chloride, 2-thiophenesulfonyl chloride,5-chloro-2-thiophenesulfonyl chloride, 2,5-dichloro-4-thiophenesulfonylchloride, 2-thiazolesulfonyl chloride, 2-methyl-4-thiazolesulfonylchloride, 1-methyl-4-imidazolesulfonyl chloride,1-methyl-4-pyrazolesulfonyl chloride,5-chloro-1,3-dimethyl-4-pyrazolesulfonyl chloride, 3-pyridinesulfonylchloride, 2-pyrimidinesulfonyl chloride and the like. If desired, asulfonyl fluoride, sulfonyl bromide or sulfonic acid anhydride may beused in place of the sulfonyl chloride in the above reaction to form theN-sulfonyl amino acid, 3.

The N-arylsulfonyl amino acid, 3, is then coupled to commerciallyavailable tyrosine esters as shown in Scheme 2 below:

where R, Ar¹, X, m and n are as defined above, R^(a) is hydrogen oralkyl but preferably is an alkyl group such as t-butyl, Z representsoptional substitution on the aryl ring and o is zero, one or two.

This coupling reaction is typically conducted using well-known couplingreagents such as carbodiimides, BOP reagent(benzotriazol-1-yloxy-tris(dimethylamino)-phosphoniumhexafluorophosphonate) and the like. Suitable carbodiimides include, byway of example, dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and the like. Ifdesired, polymer supported forms of carbodiimide coupling reagents mayalso be used including, for example, those described in TetrahedronLetters, 34(48), 7685 (1993). Additionally, well-known couplingpromoters, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole and thelike, may be used to facilitate the coupling reaction.

This coupling reaction is typically conducted by contacting theN-sulfonylamino acid, 3, with about 1 to about 2 equivalents of thecoupling reagent and at least one equivalent, preferably about 1 toabout 1.2 equivalents, of tyrosine derivative, 4, in an inert diluent,such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like. Generally, this reaction isconducted at a temperature ranging from about 0° C. to about 37° C. forabout 12 to about 24 hours. Upon completion of the reaction, thecompound 5 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like.

Alternatively, the N-sulfonyl amino acid, 3, can be converted into anacid halide which is then coupled with compound, 4, to provide compound5. The acid halide can be prepared by contacting compound 3 with aninorganic acid halide, such as thionyl chloride, phosphoroustrichloride, phosphorous tribromide or phosphorous pentachloride, orpreferably, with oxalyl chloride under conventional conditions.Generally, this reaction is conducted using about 1 to 5 molarequivalents of the inorganic acid halide or oxalyl chloride, either neator in an inert solvent, such as dichloromethane or carbon tetrachloride,at temperature in the range of about 0° C. to about 80° C. for about 1to about 48 hours. A catalyst, such as DMF, may also be used in thisreaction.

The acid halide of N-sulfonyl amino acid, 3, is then contacted with atleast one equivalent, preferably about 1.1 to about 1.5 equivalents, ofthe tyrosine derivative, 4, in an inert diluent, such asdichloromethane, at a temperature ranging from about −70° C. to about40° C. for about 1 to about 24 hours. Preferably, this reaction isconducted in the presence of a suitable base to scavenge the acidgenerated during the reaction. Suitable bases include, by way ofexample, tertiary amines, such as triethylamine, diisopropylethylamine,N-methylmorpholine and the like. Alternatively, the reaction can beconducted under Scholten-Baumann-type conditions using aqueous alkali,such as sodium hydroxide and the like. Upon completion of the reaction,compound 5 is recovered by conventional methods includingneutralization, evaporation, extraction, precipitation, chromatography,filtration, and the like.

Alternatively, compound 5 can be prepared by first forming a diaminoacid derivative and then coupling the diamino acid to the arylsulfonylhalide, 2, as shown in scheme 3 below:

where R, R^(a), Ar¹, X, Z, m, n and o are as defined above.

The diamino acid, 6, can be readily prepared by coupling amino acid, 1,with amino acid, 4, using conventional amino acid coupling techniquesand reagents, such carbodiimides, BOP reagent and the like, as describedabove. Diamino acid, 6, can then be sulfonated using sulfonyl chloride,2, and using the synthetic procedures described above to providecompound 7.

The tyrosine derivatives, 4, employed in the above reactions are eitherknown compounds or compounds that can be prepared from known compoundsby conventional synthetic procedures. For example, tyrosine derivatives,4, suitable for use in the above reactions include, but are not limitedto, L-tyrosine methyl ester, L-tyrosine t-butyl ester,L-3,5-diiodotyrosine methyl ester, L-3-iodotyrosine methyl ester,β-(4-hydroxy-naphth-1-yl)-L-alanine methyl ester,β-(6-hydroxy-naphth-2-yl)-L-alanine methyl ester, and the like. Ifdesired, of course, other esters or amides of the above-describedcompounds may also be employed.

The N-arylsulfonyl-heterocyclic amino acid-tyrosine derivative, 7, canbe used as a starting point to attach a polymer moiety at the Ar² groupby employing the process of the invention.

Amine moieties located on other portions of the molecule can be employedin the manner described above to covalently link a polymer group to themolecule. For example, amines located on Ar¹, on the heterocyclic aminoacid or on Ar² can be similarly derivatized to provide for PEGsubstitution using the process of the invention. The amine moieties canbe included in these substituents during synthesis and appropriatelyprotected as necessary. Alternatively, amine precursors can be employed.

Further, the amino substitution can be incorporated into theheterocyclic amino acid functionality and then derivatized to include apolymer moiety. For example, the heterocyclic amino acid functionalitycan be 2-carboxylpiperazine depicted in U.S. Pat. No. 6,489,300.Alternatively, commercially available 3- or 4-hydroxyproline can beoxidized to the corresponding ketone and then reductively aminated withammonia in the presence of sodium cyanoborohydride to form thecorresponding amine moiety. Still further, 4-cyanoproline can be reducedto provide for a substituted alkyl group of the formula —CH₂NH₂ whichcan be derivatized through the amine.

Still further, the amine moiety can be incorporated into the Ar²functionality. Preferably, the amine moiety is present as an amineprecursor such as a nitro or cyano group bound to Ar².

The non-derivatized compounds of Formula IIa-IIh are subsequentlycoupled to a polymer by employing the process of the invention. Scheme 4depicts an embodiment of the invention:

In Scheme 9, a PEG alcohol 100 is treated with a nucleophile 105 in thepresence of a phosphine, here PPh₃, and a diazodicarboxylate, e.g.,diisopropylazodicarboxylate, to form the protected ester 110. The esteris then hydrolyzed to form the desired conjugate 115.

The reaction occurs under mild and essentially neutral conditions. Thereaction is preferably carried out in at least one suitable solvent.Examples include halogenated solvents such as methylene chloride,aromatic solvents such as benzene or toluene, or ether solvents such astetrahydrofuran and diethylether. Other suitable solvents includeethylacetate, acetonitrile, and DMF. Most preferably, a chlorinatedsolvent or an ether solvent is employed. In a most preferred embodiment,the solvent is methylene chloride or tetrahydrofuran.

Reaction temperature is typically in the range of about −100 to 100° C.,preferably in the range of about −20 to 50° C., and even more preferablyfrom about 0° to about room temperature. In a particularly preferredembodiment, reaction temperatures are from between about −10 to 10° C.

The reaction time is in the range of about 5 minutes to about 100 hours,preferably in the range of from between about 30 minutes to about 50hours. More preferably, the reaction proceeds to completion of frombetween about 45 minutes to about 10 hours.

As mentioned above, examples of triaryl- or trialkylphosphines includetriphenylphosphine, trimethylphosphine, triethylphosphine,tributylphosphine, and 1,2-bis-(diphenylphosphino)ethane. The phosphinescan also be polymer supported or water soluble. A preferredtriarylphosphine is triphenylphosphine.

Examples of diazo compounds are diethyldiazocarboxylate,diisopropylazodicarboxylate, 4-methyl-1,2,4-triazolidine-3,5-dione,N,N,N′,N′-tetramethylazodicarboxamide, azodicarboxylic aciddipiperidide, bis(N-4-methylpiperazin-1-yl)azodicarboxamide,dimorpholinoazodicarboxamide and di-tert-butyl azodicarboxylate.

It is understood that other suitable polymeric alchohols could be usedin place of PEG and that one of ordinary skill in the art would readilybe able to modify the reaction schemes below to incorporate such otherpolymers. In some cases, the PEG moiety can be directly introduced ontothe Ar² group and, in other cases, the PEG moiety can be introduced bylinkage through a linker moiety.

Other polymers suitable for conjugation to a compound of formula IIinclude, without limitation, polyvinylpyrrolidone (PVP), polyacrylamide(PAAm), polydimethylacrylamide (PDAAm), polyvinyl alcohol (PVA),dextran, poly (L-glutamic acid) (PGA), styrene maleic anhydride (SMA),poly-N-(2-hydroxypropyl)methacrylamide (HPMA), polydivinylether maleicanhydride (DIVEMA). By way of example, PVP, PAAm and PDAAm may befunctionalized by introduction of co-monomers during radicalpolymerization. PVA and dextran each contain primary hydroxyl (OH)groups suitable for conjugation. Methods for synthesis of thesebiopolymers and for conjugating them to biological materials are wellknown in the art (see, for example, published U.S. Patent Application20040043030; U.S. Pat. No. 5,177,059; U.S. Pat. No. 6,716,821; U.S. Pat.No. 5,824,701; U.S. Pat. No. 6,664,331; U.S. Pat. No. 5,880,131; Kameda,Y. et al., Biomaterials 25: 3259-3266, 2004; Thanou, M. et al, CurrentOpinion in Investigational Drugs 4(6): 701-709, 2003; Veronese, F. M.,et al., II Farmaco 54: 497-516, 1999, all of which are incorporatedherein in their entireties).

Representative polymers suitable for use in the invention include:HO(alkylene-O)_(pp)R^(bb) mono-capped mono-hydroxy PEG (mPEG)H₂N(alkylene-O)_(pp)R^(bb) mono-capped mono-amino PEGHO(alkylene-O)_(pp)R—OH non-capped di-hydroxy PEGH₂N(alkylene-O)_(pp)R—OH non-capped mono-amino PEGHO(alkylene-O)_(pp)R^(bb) branched mono-hydroxy PEGHO(alkylene-O)_(pp)R^(bb) dendrimeric mono-hydroxy PEGwhere pp and alkylene are as defined herein and R^(bb) is preferablyselected from the group consisting of alkyl, substituted alkyl, aryl andsubstituted aryl.Other suitable polymers are shown below:

Branched PEGs:

Dendrimeric PEGs:

PEG Reagents available from SunBio (40 and 20 kDa)

These PEG polymers may be further modified by extending the chains withPEG diamines through an appropriate linker, for example a carbamate(urethane) or a urea.

A variety of nucleophilic compounds having an acidic hydrogen can beused in the process of the invention. Such compounds can be biologicallyactive compounds, i.e., therapeutic compounds (pharmaceuticals) andagricultural chemicals (pesticides, herbicides. and plant growthstimulators such as fertilizers), or food additive compounds. Examplesof groups having an acidic nitrogen that can be incorporated into suchcompounds are shown above; see the structures set forth in Tables D andD1.

Pharmaceutical Formulations

When employed as pharmaceuticals, the conjugates of this invention areusually administered in the form of pharmaceutical compositions. Theseconjugates can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular,sublingual, ophthalmic, or inhalation including administration by nasalor oral inhalation. Preferred administration routes includesubcutaneous, intravenous, and inhalation. Such compositions areprepared in a manner well known in the pharmaceutical art and compriseat least one conjugate.

The invention also provides pharmaceutical compositions comprising aconjugate according to the invention, e.g., a conjugate of Formula I, incombination with a separate compound which is an α₄β₇ inhibitor. Suchcompositions also comprise a pharmaceutically acceptable carrier orexcipient and may be administered as discussed elsewhere herein.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the conjugate of formula Itogether with pharmaceutically acceptable carriers. In making thecompositions of this invention, the active ingredient is usually mixedwith an excipient, diluted by an excipient or enclosed within such acarrier which can be in, sterile injectable solutions, and sterilepackaged powders. For subcutaneous administration, a simple carrier maycomprise a sterile solution of water, Na2HPO4, NaH2PO4, and NaCl, inproportions that provide an isotonic and physiologically acceptable pH,also know as PBS or phosphate-buffered saline. Other options are knownto those of skill in the art and include mixed solvent systems that canaffect the rate of absorption and total exposure. These options includemixed solvent systems containing glycerin, Polyethylene glycol 400, andcottonseed oil. Also of potential use are ethanol,N,N′-dimethylacetamide, propylene glycol and benzyl alcohol all of whichmay be used to manipulate permeability enhancement and hypertonicity.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

Administration of therapeutic agents by subcutaneous or intravenousformulation is well known in the pharmaceutical industry. A subcutaneousor intravenous formulation should possess certain qualities aside frombeing just a composition in which the therapeutic agent is soluble. Forexample, the formulation should promote the overall stability of theactive ingredient(s), also, the manufacture of the formulation should becost effective. All of these factors ultimately determine the overallsuccess and usefulness of an intravenous formulation.

Other accessory additives that may be included in pharmaceuticalformulations of compounds of the present invention as follow: solvents:ethanol, glycerol, propylene glycol; stabilizers: EDTA (ethylene diaminetetraacetic acid), citric acid; antimicrobial preservatives: benzylalcohol, methyl paraben, propyl paraben; buffering agents: citricacid/sodium citrate, potassium hydrogen tartrate, sodium hydrogentartrate, acetic acid/sodium acetate, maleic acid/sodium maleate, sodiumhydrogen phthalate, phosphoric acid/potassium dihydrogen phosphate,phosphoric acid/disodium hydrogen phosphate; and tonicity modifiers:sodium chloride, mannitol, dextrose.

The presence of a buffer is necessary to maintain the aqueous pH in therange of from about 4 to about 8 and more preferably in a range of fromabout 4 to about 6. The buffer system is generally a mixture of a weakacid and a soluble salt thereof, e.g., sodium citrate/citric acid; orthe monocation or dication salt of a dibasic acid, e.g., potassiumhydrogen tartrate; sodium hydrogen tartrate, phosphoric acid/potassiumdihydrogen phosphate, and phosphoric acid/disodium hydrogen phosphate.

The amount of buffer system used is dependent on (1) the desired pH; and(2) the amount of drug. Generally, the amount of buffer used is in a0.5:1 to 50:1 mole ratio of buffer:alendronate (where the moles ofbuffer are taken as the combined moles of the buffer ingredients, e.g.,sodium citrate and citric acid) of formulation to maintain a pH in therange of 4 to 8 and generally, a 1:1 to 10:1 mole ratio of buffer(combined) to drug present is used.

A useful buffer in the invention is sodium citrate/citric acid in therange of 5 to 50 mg per ml of sodium citrate to 1 to 15 mg per ml ofcitric acid, sufficient to maintain an aqueous pH of 4-6 of thecomposition.

The buffer agent may also be present to prevent the precipitation of thedrug through soluble metal complex formation with dissolved metal ions,e.g., Ca, Mg, Fe, Al, Ba, which may leach out of glass containers orrubber stoppers or be present in ordinary tap water. The agent may actas a competitive complexing agent with the drug and produce a solublemetal complex leading to the presence of undesirable particulates.

In addition, the presence of an agent, e.g., sodium chloride in anamount of about of 1-8 mg/ml, to adjust the tonicity to the same valueof human blood may be required to avoid the swelling or shrinkage oferythrocytes upon administration of the intravenous formulation leadingto undesirable side effects such as nausea or diarrhea and possibly toassociated blood disorders. In general, the tonicity of the formulationmatches that of human blood which is in the range of 282 to 288 mOsm/kg,and in general is 285 mOsm/kg , which is equivalent to the osmoticpressure corresponding to a 0.9% solution of sodium chloride.

The intravenous formulation can be administered by direct intravenousinjection, i.v. bolus, or can be administered by infusion by addition toan appropriate infusion solution such as 0.9% sodium chloride injectionor other compatible infusion solution.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The conjugate is effective over a wide dosage range and is generallyadministered in a pharmaceutically effective amount. It, will beunderstood, however, that the amount of the conjugate actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be breathed directly from thenebulizing device or the nebulizing device may be attached to a facemasks tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner. For inhalation or insufflation administration,it is preferred that the total molecular weight of the conugate isbetween about 10,000 Daltons and 70,000 Daltons, more preferably betweenabout 20,000 Daltonsand 45,000 Daltons.

Polymer Conjugates

Compounds of this invention as formulated and administered are polymerconjugates. Polymer conjugates are anticipated to provide benefits overnon-conjugated polymers, such as improved solubility and in vivostability.

As such, single polymer molecule may be employed for conjugation withthe compounds of the present invention, although it is also contemplatedthat more than one polymer molecule can be attached as well, typicallythrough a carrier. The conjugated compounds of the present invention mayfind utility in both in vivo as well as non-in vivo applications.Additionally, it will be recognized that the conjugating polymer mayutilize any other groups, moieties, or other conjugated species, asappropriate to the end use application. As an example, it may beadvantageous in some applications to functionalize the polymer to renderit reactive and enable it to conjugate to a compound of formula II andto enhance various properties or characteristics of the overallconjugated material. Accordingly, the polymer may contain anyfunctionality, repeating groups, linkages, or other constituentstructures which do not preclude the efficacy of the conjugatedcompounds of the present invention for its intended purpose.

Illustrative polymers that are usefully employed to achieve thesedesirable characteristics are described supra, as well as in PCT WO01/54690 (to Zheng et al.) incorporated by reference herein in itsentirety. The polymer may be coupled to the compounds of the presentinvention (preferably via a linker moiety) to form stable bonds that arenot significantly cleavable by human enzymes. Generally, for a bond tobe not ‘significantly’ cleavable requires that no more than about 20% ofthe bonds connecting the polymer and the compounds of the presentinvention to which the polymer is linked, are cleaved within a 24 hourperiod, as measured by standard techniques in the art including, but notlimited to, high pressure liquid chromatography (HPLC).

Generally, the compounds of this invention contain at least about 2compounds of formula II bound to a polymer. The final amount is abalance between maximizing the extent of the reaction while minimizingnon-specific modifications of the product and, at the same time,defining chemistries that will maintain optimum activity, while at thesame time optimizing the half-life of the compounds of the presentinvention. Preferably, at least about 50% of the biological activity ofthe compounds of the present invention is retained, and most preferably100% is retained.

As noted above in the preferred practice of the present invention,polyalkylene glycol residues of C₂-C₄ alkyl polyalkylene glycols,preferably polyethylene glycol (PEG), or poly(oxy)alkylene glycolresidues of such glycols are advantageously incorporated in the polymersystems of interest. Thus, the polymer to which the compounds of thepresent invention are attached may be a homopolymer of polyethyleneglycol (PEG) or is a polyoxyethylated polyol, provided in all cases thatthe polymer is soluble in water at room temperature. Non-limitingexamples of such polymers include polyalkylene oxide homopolymers suchas PEG or polypropylene glycols, polyoxyethylenated glycols, copolymersthereof and block copolymers thereof, provided that the water solubilityof the block copolymer is maintained.

Examples of polyoxyethylated polyols include, but are not limited to,polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylatedglucose, or the like. The glycerol backbone of polyoxyethylated glycerolis the same backbone occurring naturally in, for example, animals andhumans in mono-, di-, and triglycerides. Therefore, this branching wouldnot necessarily be seen as a foreign agent in the body.

Those of ordinary skill in the art will recognize that the foregoinglist is merely illustrative and that all polymer materials having thequalities described herein are contemplated. The polymer need not haveany particular molecular weight, but it is preferred that the molecularweight be between about 100 and 100,000, preferably from about 10,000 to80,000; more preferably from about 20,000 to about 70,000. Inparticular, sizes of 20,000 or more are most effective at preventingloss of the product due to filtration in the kidneys.

By PEG derivative is meant a polyethylene glycol polymer in which one orboth of the terminal hydroxyl groups found in polyethylene glycol itselfhas been modified. Examples of suitable modifications include replacingone or both hydroxyl group(s) with alternative functional groups, whichmay be protected or unprotected, with low molecular weight ligands, orwith another macromolecule or polymer. Modification of the terminalhydroxyl groups in the polyethylene glycol may be achieved by reactingthe polyethylene glycol with compounds comprising complementary reactivefunctional groups, including functional groups which are able to undergoa reaction with the hydroxyl groups in polyethylene glycol. The PEGderivatives of the compounds of this invention may contain one or morepolyethylene glycol (PEG) substituents covalently attached thereto by alinking group.

The following formulation examples illustrate the pharmaceuticalcompositions of the present invention.

FORMULATION EXAMPLE 1

Hard gelatin capsules containing the following ingredients are prepared:Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

FORMULATION EXAMPLE 2

A tablet formula is prepared using the ingredients below: QuantityIngredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

FORMULATION EXAMPLE 3

A dry powder inhaler formulation is prepared containing the followingcomponents: Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

FORMULATION EXAMPLE 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows: Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mgStarch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone4.0 mg (as 10% solution in sterile water) Sodium carboxymethyl starch4.5 mg Magnesium stearate 0.5 mg Talc 1.0 mg Total 120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° C. to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

FORMULATION EXAMPLE 5

Capsules, each containing 40 mg of medicament are made as follows:Quantity Ingredient (mg/capsule) Active Ingredient 40.0 mg Starch 109.0mg Magnesium stearate 1.0 mg Total 150.0 mg

The active ingredient, starch and magnesium stearate are blended, passedthrough a No. 20 mesh U.S. sieve, and filled into hard gelatin capsulesin 150 mg quantities.

FORMULATION EXAMPLE 6

Suppositories, each containing 25 mg of active ingredient are made asfollows: Ingredient Amount Active Ingredient 25 mg Saturated fatty acidglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

FORMULATION EXAMPLE 7

Suspensions, each containing 50 mg of medicament per 5.0 ml dose aremade as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthan gum4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose(89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Colorq.v. Purified water to 5.0 ml

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

FORMULATION EXAMPLE 8

Quantity Ingredient (mg/capsule) Active Ingredient 15.0 mg Starch 407.0mg Magnesium stearate 3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

FORMULATION EXAMPLE 9

A subcutaneous formulation may be prepared as follows: IngredientQuantity Active Ingredient 50 mg · mL mg Phosphate buffered saline 1.0ml

FORMULATION EXAMPLE 10

A topical formulation may be prepared as follows: Ingredient QuantityActive Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g WhiteSoft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

FORMULATION EXAMPLE 11

An intravenous formulation may be prepared as follows: IngredientQuantity Active Ingredient 250 mg Isotonic saline 100 ml

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce thepharmaceutical composition to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472 which is herein incorporated byreference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985).

As noted above, the compounds described herein are suitable for use in avariety of drug delivery systems described above. Additionally, in orderto enhance the in vivo serum half-life of the administered compound, thecompounds may be encapsulated, introduced into the lumen of liposomes,prepared as a colloid, or other conventional techniques may be employedwhich provide an extended serum half-life of the compounds. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each ofwhich is incorporated herein by reference.

Utility

The conjugates of this invention are VLA-4 antagonists. Some also haveat least a partial affinity for alpha4 beta7 integrins. The conjugatesprovide enhanced in vivo retention as compared to the non-conjugatedcompounds. The improved retention of the conjugate within the bodyresults in lower required dosages of the drug, which in turn results infewer side effects and reduced likelihood of toxicity. In addition, thedrug formulation may be administered less frequently to the patientwhile achieving a similar or improved therapeutic effect.

The conjugates of this invention have improved inhibition, in vivo, ofadhesion of leukocytes to endothelial cells mediated by inhibition ofalpha4 beta1 or alpha4 beta7binding to cellular receptors such asVCAM-1. fibronectin and MadCAM. Preferably, the conjugates of thisinvention can be used, e.g., by infusion, or by subcutaneous injectionor oral administration, for the treatment of diseases mediated by alpha4beta1 or alpha4 beta7 or, in general terms, leucocyte adhesion. Theconjugates of the invention can be used to treat a variety ofinflammatory brain disorders, especially central nervous systemdisorders in which the endothelium/leukocyte adhesion mechanism resultsin destruction to otherwise healthy brain tissue. Thus, the conjugatesof the invention can be used for, e.g., the treatment of experimentalautoimmune encephalomyelitis (EAE), multiple sclerosis (MS), meningitis,and encephalitis.

The conjugates of the invention can also be used to treat disorders anddiseases due to tissue damage in other organ systems, i.e., where tissuedamage also occurs via an adhesion mechanism resulting in migration oractivation of leukocytes. Examples of such diseases in mammalianpatients are inflammatory diseases such as asthma, Alzheimer's disease,atherosclerosis, AIDS dementia, diabetes (including acute juvenile onsetdiabetes), inflammatory bowel disease (including ulcerative colitis andCrohn's disease), rheumatoid arthritis, tissue transplantationrejection, tumor metastasis, stroke, and other cerebral traumas,nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemiaand acute leukocyte-mediated lung injury such as that which occurs inadult respiratory distress syndrome.

Still other disease conditions which may be treated using conjugates ofthe invention include erythema nodosum, allergic conjunctivitis, opticneuritis, uveitis, allergic rhinitis, ankylosing spondylitis, psoriaticarthritis, vasculitis, Reiter's syndrome, systemic lupus erythematosus,progressive systemic sclerosis, polymyositis, dermatomyositis, Wegner'sgranulomatosis, aortitis, sarcoidosis, lymphocytopenia, temporalarteritis, pericarditis, myocarditis, congestive heart failure,polyarteritis nodosa, hypersensitivity syndromes, allergy,hypereosinophilic syndromes, Churg-Strauss syndrome, chronic obstructivepulmonary disease, hypersensitivity pneumonitis, chronic activehepatitis, interstitial cystitis, autoimmune endocrine failure, primarybiliary cirrhosis, autoimmune aplastic anemia, chronic persistenthepatitis and thyroiditis.

The invention also provides methods for treating a disease state causedor exacerbated at least in part by alpha 4 integrin-mediated lekocytebinding in a patient, which methods comprise co-administration of aneffective amount of a conjugate of the invention, e.g., a conjugate ofFormula I, and an effective amount of a separate compound which is anα₄β₇ inhibitor. The co-adminstration can be carried out simultaneouslyor sequentially. For example, administration of the conjugate of theinvention can precede adminstration of the α₄β₇ inhibitor by minutes orhours. Alternatively, the α₄β₇ inhibitor can be administered prior tothe conjugate of the invention.

Appropriate in vivo models for demonstrating efficacy in treatinginflammatory responses include EAE (experimental autoimmuneencephalomyelitis) in mice, rats, guinea pigs or primates, as well asother inflammatory models dependent upon α4 integrins.

Inflammatory bowel disease is a collective term for two similar diseasesreferred to as Crohn's disease and ulcerative colitis. Crohn's diseaseis an idiopathic, chronic ulceroconstrictive inflammatory diseasecharacterized by sharply delimited and typically transmural involvementof all layers of the bowel wall by a granulomatous inflammatoryreaction. Any segment of the gastrointestinal tract, from the mouth tothe anus, may be involved, although the disease most commonly affectsthe terminal ileum and/or colon. Ulcerative colitis is an inflammatoryresponse limited largely to the colonic mucosa and submucosa.Lymphocytes and macrophages are numerous in lesions of inflammatorybowel disease and may contribute to inflammatory injury.

Asthma is a disease characterized by increased responsiveness of thetracheobronchial tree to various stimuli potentiating paroxysmalconstriction of the bronchial airways. The stimuli cause release ofvarious mediators of inflammation from IgE-coated mast cells includinghistamine, eosinophilic and neutrophilic chemotactic factors,leukotrines, prostaglandin and platelet activating factor. Release ofthese factors recruits basophils, eosinophils and neutrophils, whichcause inflammatory injury.

Atherosclerosis is a disease of arteries (e.g., coronary, carotid, aortaand iliac). The basic lesion, the atheroma, consists of a raised focalplaque within the intima, having a core of lipid and a covering fibrouscap. Atheromas compromise arterial blood flow and weaken affectedarteries. Myocardial and cerebral infarcts are a major consequence ofthis disease. Macrophages and leukocytes are recruited to atheromas andcontribute to inflammatory injury.

Rheumatoid arthritis is a chronic, relapsing inflammatory disease thatprimarily causes impairment and destruction of joints. Rheumatoidarthritis usually first affects the small joints of the hands and feetbut then may involve the wrists, elbows, ankles and knees. The arthritisresults from interaction of synovial cells with leukocytes thatinfiltrate from the circulation into the synovial lining of the joints.See e.g., Paul, Immunology (3d ed., Raven Press, 1993).

Another indication for the conjugates of this invention is in treatmentof organ or graft rejection mediated by VLA-4. Over recent years therehas been a considerable improvement in the efficiency of surgicaltechniques for transplanting tissues and organs such as skin, kidney,liver, heart, lung, pancreas and bone marrow. Perhaps the principaloutstanding problem is the lack of satisfactory agents for inducingimmunotolerance in the recipient to the transplanted allograft or organ.When allogeneic cells or organs are transplanted into a host (i.e., thedonor and donee are different individuals from the same species), thehost immune system is likely to mount an immune response to foreignantigens in the transplant (host-versus-graft disease) leading todestruction of the transplanted tissue. CD8⁺ cells, CD4 cells andmonocytes are all involved in the rejection of transplant tissues.Conjugates of this invention which bind to alpha-4 integrin are useful,inter alia, to block alloantigen-induced immune responses in the doneethereby preventing such cells from participating in the destruction ofthe transplanted tissue or organ. See, e.g., Paul et al., TransplantInternational 9, 420-425 (1996); Georczynski et al., Immunology 87,573-580 (1996); Georcyznski et al., Transplant. Immunol. 3, 55-61(1995); Yang et al., Transplantation 60, 71-76 (1995); Anderson et al.,APMIS 102, 23-27 (1994).

A related use for conjugates of this invention which bind to VLA-4 is inmodulating the immune response involved in “graft versus host” disease(GVHD). See e.g., Schlegel et al., J. Immunol. 155, 3856-3865 (1995).GVHD is a potentially fatal disease that occurs when immunologicallycompetent cells are transferred to an allogeneic recipient. In thissituation, the donor's immunocompetent cells may attack tissues in therecipient. Tissues of the skin, gut epithelia and liver are frequenttargets and may be destroyed during the course of GVHD. The diseasepresents an especially severe problem when immune tissue is beingtransplanted, such as in bone marrow transplantation; but less severeGVHD has also been reported in other cases as well, including heart andliver transplants. The therapeutic agents of the present invention areused, inter alia, to block activation of the donor T-cells therebyinterfering with their ability to lyse target cells in the host.

The formulations of the present invention are especially useful in thetreatment of multiple sclerosis, rheumatoid arthritis and asthma.

A further use of the conjugates of this invention is inhibiting tumormetastasis. Several tumor cells have been reported to express VLA-4 andcompounds which bind VLA-4 block adhesion of such cells to endothelialcells. Steinback et al., Urol. Res. 23, 175-83 (1995); Orosz et al.,Int. J. Cancer 60, 867-71 (1995); Freedman et al., Leuk. Lymphoma 13,47-52 (1994); Okahara et al., Cancer Res. 54, 3233-6 (1994).

Compounds having the desired biological activity may be modified asnecessary to provide desired properties such as improved pharmacologicalproperties (e.g., in vivo stability, bio-availability), or the abilityto be detected in diagnostic applications. Stability can be assayed in avariety of ways such as by measuring the half-life of the proteinsduring incubation with peptidases or human plasma or serum. A number ofsuch protein stability assays have been described (see, e.g., Verhoef etal., Eur. J. Drug Metab. Pharmacokinet., 1990, 15(2):83-93).

A further use of the conjugates of this invention is in treatingmultiple sclerosis. Multiple sclerosis is a progressive neurologicalautoimmune disease that affects an estimated 250,000 to 350,000 peoplein the United States. Multiple sclerosis is thought to be the result ofa specific autoimmune reaction in which certain leukocytes attack andinitiate the destruction of myelin, the insulating sheath covering nervefibers. In an animal model for multiple sclerosis, murine monoclonalantibodies directed against VLA-4 have been shown to block the adhesionof leukocytes to the endothelium, and thus prevent inflammation of thecentral nervous system and subsequent paralysis in the animals¹⁶.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985).

The amount administered to the patient will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis or therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, compositionsare administered to a patient already suffering from a disease in anamount sufficient to cure or at least partially arrest the symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as “therapeutically effective dose.” Amounts effective forthis use will depend on the disease condition being treated as well asby the judgment of the attending clinician depending upon factors suchas the severity of the inflammation, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient are in the form ofpharmaceutical compositions described above. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration.

The therapeutic dosage of the conjugates of the present invention willvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the conjugate, thehealth and condition of the patient, and the judgment of the prescribingphysician. For example, for intravenous administration, the dose willtypically be in the range of about 20 μg to about 2000 μg per kilogrambody weight, preferably about 20 μg to about 500 μg, more preferablyabout 100 μg to about 300 μg per kilogram body weight. Suitable dosageranges for intranasal administration are generally about 0.1 μg to 1 mgper kilogram body weight. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Conjugates of this invention are also capable of binding or antagonizingthe actions of α₆β₁, α₉β₁, α₄β₇, α_(d)β₂, α_(e)β₇ integrins (althoughα₄β₁ and α₉β₁ are preferred in this invention). Accordingly, conjugatesof this invention are also useful for preventing or reversing thesymptoms, disorders or diseases induced by the binding of theseintegrins to their respective ligands.

For example, International Publication Number WO 98/53817, publishedDec. 3, 1998 (the disclosure of which is incorporated herein byreference in its entirety) and references cited therein describedisorders mediated by α₄β₇. This reference also describes an assay fordetermining antagonism of α₄β₇ dependent binding to VCAM-Ig fusionprotein.

Additionally, compounds that bind α_(d)β₂ and α_(e)β₇ integrins areparticularly useful for the treatment of asthma and related lungdiseases. See, for example, M. H. Grayson et al., J. Exp. Med. 1998,188(11) 2187-2191. Compounds that bind α_(e)β₇ integrin are also usefulfor the treatment of systemic lupus erythematosus (see, for example, M.Pang et al., Arthritis Rheum. 1998, 41(8),1456-1463); Crohn's disease,ulcerative colitis and inflammatory bowel disease (IBD) (see, forexample, D. Elewaut et al., Scand J. Gastroenterol 1998, 33(7) 743-748);Sjogren's syndrome (see, for example, U. Kroneld et al., Scand J.Gastroenterol 1998, 27(3), 215-218); and rheumatoid arthritis (see, forexample, Scand J. Gastroenterol 1996, 44(3), 293-298). And compoundsthat bind α₆β₁ may be useful in preventing fertilization (see, forexample, H. Chen et al., Chem. Biol. 1999, 6, 1-10).

In another aspect of the invention, the conjugates and compositionsdescribed herein can be used to inhibit immune cell migration from thebloodstream to the central nervous system in the instance of, forexample, multiple sclerosis, or to areas which result ininflammatory-induced destruction of the myelin. Preferably, thesereagents inhibit immune cell migration in a manner that inhibitsdemyelination and that further may promote remyelination. The reagentsmay also prevent demyelination and promote remyelination of the centralnervous system for congenital metabolic disorders in which infiltratingimmune cells affect the development myelin sheath, mainly in the CNS.The reagents preferably also reduce paralysis when administrered to asubject with paralysis induced by a demyelinating disease or condition.

Inflammatory diseases that are included for treatment by thecompositions, conjugates and methods disclosed herein include generallyconditions relating to demyelination. Histologically, myelinabnormalities are either demyelinating or dysmyelinating. Demyelinationimplies the destruction of myelin. Dysmyelination refers to defectiveformation or maintenance of myelin resulting from dysfunction of theoligodendrocytes. Preferably, the compositions and methods disclosedherein are contemplated to treat diseases and conditions relating todemyelination and aid with remyelination. Additional diseases orconditions contemplated for treatment include meningitis, encephalitis,and spinal cord injuries and conditions generally which inducedemyelination as a result of an inflammatory response. The conjugates,compositions and methods disclosed herein are not directed towardsdiseases and conditions wherein there is, for example, a genetic defectleading to improper myelin formation, e.g., dysmyelination.

The compositions, conjugates and cocktails disclosed herein arecontemplated for use in treating conditions and diseases associated withdemyelination. Diseases and conditions involving demyelination include,but are not limited to, multiple sclerosis, congenital metabolicdisorders (e.g., phenylketonuria, Tay-Sachs disease, Niemann-Pickdisease, Gaucher's disease, Hurler's syndrome, Krabbe's disease andother leukodystrophies), neuropathies with abnormal myelination (e.g.,Guillain Barré, chronic immune demyelinating polyneuropathy (CIDP),multifocal CIDP, anti-MAG syndrome, GALOP syndrome, anti-sulfatideantibody syndrome, anti-GM2 antibody syndrome, POEMS syndrome,perineuritis, IgM anti-GD1b antibody syndrome), drug relateddemyelination (e.g., caused by the administration of chloroquine, FK506,perhexiline, procainamide, and zimeldine), other hereditarydemyelinating conditions (e.g., carbohydrate-deficient glycoprotein,Cockayne's syndrome, congenital hypomyelinating, congenital musculardystrophy, Farber's disease, Marinesco-Sjögren syndrome, metachromaticleukodystrophy, Pelizaeus-Merzbacher disease, Refsum disease, prionrelated conditions, and Salla disease) and other demyelinatingconditions (e.g., meningitis, encephalitis or spinal cord injury) ordiseases.

There are various disease models that can be used to study thesediseases in vivo. For example, animal models include but are not limitedto: TABLE III Disease Model Species EAE Mouse, rat, guinea pigMyelin-oligodendrocyte glycoprotein (MOG) Rat induced EAE TNF-αtransgenic model of demyelination MouseMultiple Sclerosis

The most common demyelinating disease is multiple sclerosis, but manyother metabolic and inflammatory disorders result in deficient orabnormal myelination. MS is a chronic neurologic disease, which appearsin early adulthood and progresses to a significant disability in mostcases. There are approximately 350,000 cases of MS in the United Statesalone. Outside of trauma, MS is the most frequent cause of neurologicdisability in early to middle adulthood.

The cause of MS is yet to be determined. MS is characterized by chronicinflammation, demyelination and gliosis (scarring). Demyelination mayresult in either negative or positive effects on axonal conduction.Positive conduction abnormalities include slowed axonal conduction,variable conduction block that occurs in the presence of high-but notlow-frequency trains of impulses or complete conduction block. Positiveconduction abnormalities include ectopic impulse generation,spontaneously or following mechanical stress and abnormal “cross-talk”between demyelinated exons.

T cells reactive against myelin proteins, either myelin basic protein(MBP) or myelin proteolipid protein (PLP) have been observed to mediateCNS inflammation in experimental allergic encephalomyelitis. Patientshave also been observed as having elevated levels of CNS immunoglobulin(Ig). It is further possible that some of the tissue damage observed inMS is mediated by cytokine products of activated T cells, macrophages orastrocytes.

Today, 80% patients diagnosed with MS live 20 years after onset ofillness. Therapies for managing MS include: (1) treatment aimed atmodification of the disease course, including treatment of acuteexacerbation and directed to long-term suppression of the disease; (2)treatment of the symptoms of MS; (3) prevention and treatment of medicalcomplications; and (4) management of secondary personal and socialproblems.

The onset of MS may be dramatic or so mild as to not cause a patient toseek medical attention. The most common symptoms include weakness in oneor more limbs, visual blurring due to optic neuritis, sensorydisturbances, diplopia and ataxia. The course of disease may bestratified into three general categories: (1) relapsing MS, (2) chronicprogressive MS, and (3) inactive MS. Relapsing MS is characterized byrecurrent attacks of neurologic dysfunction. MS attacks generally evolveover days to weeks and may be followed by complete, partial or norecovery. Recovery from attacks generally occurs within weeks to severalmonths from the peak of symptoms, although rarely some recovery maycontinue for 2 or more years.

Chronic progressive MS results in gradually progressive worseningwithout periods of stabilization or remission. This form develops inpatients with a prior history of relapsing MS, although in 20% ofpatients, no relapses can be recalled. Acute relapses also may occurduring the progressive course.

A third form is inactive MS. Inactive MS is characterized by fixedneurologic deficits of variable magnitude. Most patients with inactiveMS have an earlier history of relapsing MS.

Disease course is also dependent on the age of the patient. For example,favourable prognostic factors include early onset (excluding childhood),a relapsing course and little residual disability 5 years after onset.By contrast, poor prognosis is associated with a late age of onset(i.e., age 40 or older) and a progressive course. These variables areinterdependent, since chronic progressive MS tends to begin at a laterage that relapsing MS. Disability from chronic progressive MS is usuallydue to progressive paraplegia or quadriplegia (paralysis) in patients.In one aspect of the invention, patients will preferably be treated whenthe patient is in remission rather then in a relapsing stage of thedisease.

Short-term use of either adrenocorticotropic hormone or oralcorticosteroids (e.g., oral prednisone or intravenousmethylprednisolone) is the only specific therapeutic measure fortreating patients with acute exacerbation of MS.

Newer therapies for MS include treating the patient with interferonbeta-1b, interferon beta-1a, and Copaxone® (formerly known as copolymer1). These three drugs have been shown to significantly reduce therelapse rate of the disease. These drugs are self-administeredintramuscularly or subcutaneously.

However, none of the current treatment modalities inhibit demyelination,let alone promotes or allows spontaneous remyelination or reducesparalysis. One aspect of the invention contemplates treating MS withagents disclosed herein either alone or in combination with otherstandard treatment modalities.

Congenital Metabolic Disorders

Congenital metabolic disorders include phenylketonuria (PKU) and otheraminoacidurias, Tay-Sachs disease, Niemann-Pick disease, Gaucher'sdisease, Hurler's syndrome, Krabbe's disease and other leukodystrophiesthat impact the developing sheath as described more fully below.

PKU is an inherited error of metabolism caused by a deficiency in theenzyme phenylalanine hydroxylase. Loss of this enzyme results in mentalretardation, organ damage, unusual posture and can, in cases of maternalPKU, severely compromise pregnancy. A model for studying PKU has beendiscovered in mice. Preferably infants identified with PKU are sustainedon a phenylalanine free or lowered diet. An aspect of the inventionwould be to combine such diets with the conjugates and compositionsdisclosed herein to prevent demyelination and remyelinate cells damageddue to PKU.

Classical Tay-Sachs disease appears in the subject at about age 6 monthsand will eventually result in the death of the subject by age 5 years.The disease is due to the lack of the enzyme, hexoaminidase A (hex A),which is necessary for degrading certain fatty substances in the brainand nerve cells. The substances in the absence of the enzyme accumulateand lead to the destruction of nerve cells. Another form of hex A enzymedeficiency occurs later in life and is referred to as juvenile, chronicand adult onset forms of hex A deficiency. Symptoms are similar to thosethat characterize classical Tay-Sachs disease. There is also an adultonset form of the enzyme deficiency. Currently there is no cure ortreatment for the disease/deficiency, only the preventative measure ofin utero testing of the fetus for the disease. Thus, the conjugates andcompositions disclosed herein may be useful in ameliorating orpreventing the destruction of nerve cells in such patients.

Niemann-Pick disease falls into three categories: the acute infantileform, Type B is a less common, chronic, non-neurological form, and TypeC is a biochemically and genetically distinct form of the disease. In anormal individual, cellular cholesterol is imported into lysosomes forprocessing, after which it is released. Cells taken from subjects withNiemann-Pick have been shown to be defective in releasing cholesterolfrom lysosomes. This leads to an excessive build-up of cholesterolinside lysosomes, causing processing errors. NPC1 was found to haveknown sterol-sensing regions similar to those in other proteins, whichsuggests it plays a role in regulating cholesterol traffic. Nosuccessful therapies have been identified for Types A and C forms ofNeumann-Pick. For Type C, patients are recommended to follow alow-cholesterol diet. Thus, the conjugates and compositions disclosedherein may be useful in ameliorating or preventing the destruction ofthe cells.

Gaucher's disease is an inherited illness caused by a gene mutation.Normally, this gene is responsible for an enzyme calledglucocerebrosidase that the body needs to break down the fat,glucocerebroside. In patients with Gaucher's disease, the body is notable to properly produce this enzyme and the fat cannot be broken down.Like Tay-Sachs disease, Gaucher's disease is considerably more common inthe descendants of Jewish people from Eastern Europe (Ashkenazi),although individuals from any ethnic group may be affected. Among theAshkenazi Jewish population, Gaucher's disease is the most commongenetic disorder, with an incidence of approximately 1 in 450 persons.In the general public, Gaucher's disease affects approximately 1 in100,000 persons.

In 1991, enzyme replacement therapy became available as the firsteffective treatment for Gaucher's disease. The treatment consists of amodified form of the glucocerebrosidase enzyme given intravenously. Itis contemplated that the compositions and conjugates disclosed hereincan be used alone or more preferably in combination withglycocerebrosidase administration to treat the disease in an afflictedsubject.

Hurler's syndrome, also known as mucopolysaccharidosis type I, is aclass of overlapping diseases. These genetic diseases share in commonthe cellular accumulation of mucopolysaccharides in fibroblasts. Thediseases are genetically distinguishable. Fibroblast and bone marrowtransplantation does not seem to be helpful, thus conjugates andcompositions useful in ameliorating disease severity and progression areneeded. The conjugates and compositions disclosed herein may beadministered to a subject to ameliorate disease progression and/orseverity.

Krabbe's disease (also known as Globoid cell leukodystrophy) is anautosomal recessive condition resulting from galactosylceramidase (orgalactocerebrosidase) deficiency, a lysosomal enzyme that catabolises amajor lipid component of myelin. Incidence in France is an estimated1:150,000 births. The disease leads to demyelination of the central andperipheral nervous system. Onset generally occurs during the first yearof life and the condition is rapidly progressive, but juvenile,adolescent or adult onset forms have also been reported, with a morevariable rate of progression. Diagnosis is established from enzyme assay(galactosylceramidase deficiency). There are several natural animalmodels (mouse, dog, monkey). Krabbe's disease, like allleukodystrophies, has no known cures or effective treatments. Oneembodiment of the instant invention is to use the compositions andconjugates disclosed herein to treat or ameliorate Krabbe's disease andother leukodystrophies.

Leukodystrophies are a group of genetically determined progressivedisorders that affect the brain, spinal cord and peripheral nerves. Theyinclude adrenoleukodystrophy (ALD), adrenomyeloneuropathy (AMN),Aicardi-Goutiers syndrome, Alexander's disease, CACH (i.e., childhoodataxia with central nervous system hypomyelination or vanishing whitematter disease), CADASIL (i.e., cerebral autosomal dominant arteriopathywith subcortical infarcts and leukoencephalopathy), Canavan disease(spongy degeneration), Cerebrotendinous Xanthomatosis (CTX), Krabbe'sdisease (discussed above), metachromatic leukodystrophy (MLD), neonataladrenoleukodystrophy, ovarioleukodystrophy syndrome,Pelizaeus-Merzbacher disease (X-linked spastic paraglegia), Refsumdisease, van der Knaap syndrome (vaculating leukodystrophy withsubcortical cysts) and Zellweger syndrome. None of the diseases haveeffective treatments let alone cures. Consequently, means of treating orameliorating the symptoms of the disease, such as by using thecompositions and conjugates disclosed herein, is needed.

Neuropathies with Abnormal Myelination

A variety of chronic immune polyneuropathies exist which result indemyelination in the patient. The age of onset for the conditions variesby condition. Standard treatments for these diseases exist and could becombined with the compositions and conjugates disclosed herein.Alternatively, the compositions and conjugates disclosed can be usedalone. Existing standard therapies include the following: TABLE IVNeuropathy Clinical Features Treatment Chronic Immune Onset between 1-80years. T-cell immunosuppression Demyelinating Characterized by weakness,with prednisone, Polyneuropathy (CIDP) sensory loss, and nervecyclosporine A or hypertrophy. methotrexate, HIG, plasma exchangeMultifocal CIDP Onset between 28 to 58 years T cell immunosuppressionand characterized by with prednisone asymmetric weakness, Humanimmunoglobulin (HIG) sensory loss with a course that is slowlyprogressive or relapsing-remitting. Multifocal Motor Onset ranges from25 to 70 HIG Neuropathy (MMN) years, with twice as many B cellimmunosuppression men as women. Features with plasma exchange includeweakness, muscle cyclophosphamide, atrophy, fasciculations, and Rituxancramps which are progressive over 1-30 years. Neuropathy with IgM Onsetis usually over age 50 B-cell immunosuppression binding to Myelin- andis characterized by plasma exchange Associated Glycoprotein sensory loss(100%), cyclophosphamide (MAG) weakness, gain disorder, Rituxan tremorwhich is all slowly α-interferon progressive. cladribine or fludarabineprednisone GALOP Syndrome (Gait A gait disorder with HIG disorder,Autoantibody, polyneuropathy Plasma exchange Late-age, Onset,cyclophosphamide Polyneuropathy) POEMS Syndrome Onset occurs between 27and Osteosclerotic lesions are (Polyneuropathy, 80 years with weakness,treated with irradiation. Organomegaly, sensory loss, reduced orWidespread lesions with Endocrinopathy, M- absent tendon reflexes, skinchemotherapy (Melphalan Protein and Skin disorders and other features.and prednisone). changes) also known as Crow-Fukase Syndrome andTakatsuki diseaseDrug and Radiation Induced Demyelination

Certain drugs and radiation can induce demyelination in subjects. Drugsthat are responsible for demyelination include but are not limited tochloroquine, FK506, perhexiline, procainamide, and zimeldine.

Radiation also can induce demyelination. Central nervous system (CNS)toxicity due to radiation is believed to be cause by (1) damage tovessel structures, (2) deletion of oligodendrocyte-2 astrocyteprogenitors and mature oligodendrocytes, (3) deletion of neural stemcell populations in the hippocampus, cerebellum and cortex, andgeneralized alterations of cytokine expression. Most radiation damageresults from radiotherapies administered during the treatment of certaincancers. See for review Belka et al., 2001 Br. J. Cancer 85: 1233-9.However, radiation exposure may also be an issue for astronauts(Hopewell, 1994 Adv. Space Res. 14: 433-42) as well as in the event ofexposure to radioactive substances.

Patients who have received drugs or been exposed accidentally orintentionally to radiation may experience a benefit by administered oneof the conjugates or compositions disclosed herein to preventdemyelination or to promote remyelination.

Conditions Involving Demyelination

Additional inherited syndromes/diseases that result in demyelinationinclude Cockayne's syndrome, congenital hypomyelinating, Farber'sdisease, metachromatic leukodystrophy, Peliszaeus-Merzbacher disease,Refsum, prion related conditions and Salla disease.

Cockayne's syndrome (CS) is a rare inherited disorder in which peopleare sensitive to sunlight, have short stature and have the appearance ofpremature aging. In the classical form of Cockayne's syndrome (Type I),the symptoms are progressive and typically become apparent after the ageof one year. An early onset or congenital form of Cockayne's syndrome(Type II) is apparent at birth. Interestingly, unlike other DNA repairdiseases, Cockayne's syndrome is not linked to cancer. CS is amulti-system disorder that causes both profound growth failure of thesoma and brain and progressive cachexia, retinal, cochlear, andneurologic degeneration, with a leukodystrophy and demyelinatingneuropathy without an increase in cancer. After exposure to UV (e.g.,sunlight), subjects with Cockayne's syndrome can no longer performtranscription-coupled repair. Two genes defective in Cockayne'ssyndrome, CSA and CSB, have been identified so far. The CSA gene isfound on chromosome 5. Both genes code for proteins that interacts withcomponents of the transcriptional machinery and with DNA repairproteins.

To date, no cures or effective treatments for patients with this diseasehave been identified. Thus, one aspect of the invention is treatment ofthis disease with the conjugates and compositions disclosed herein.

Congenital hypomyelination has several names including congenitaldysmyelinating neuropathy, congenital hypomyelinating polyneuropathy,congenital hypomyelination (Onion Bulb) polyneuropathy, congenitalhypomyelination neuropathy, congenital neuropathy caused byhypomyelination, hypomyelination neuropathy and CHN. Hereditaryperipheral neuropathies, among the most common genetic disorders inhumans, are a complex, clinically and genetically heterogeneous group ofdisorders that produce progressive deterioration of the peripheralnerves. Congenital hypomyelination is one of a group of disorders. Thisgroup includes hereditary neuropathy with liability to pressure palsies,Charcot-Marie-Tooth disease, Dejerine-Sottas syndrome, and congenitalhypomyelinating neuropathy. There are no known cures or effectivetreatments for any of these disorders.

Farber's disease has several names including: Farber lipogranulomatosis,ceremidase deficiency, acid ceramidase deficiency, AC deficiency,N-laurylsphingosine deacylase deficiency, and N-acylsphingosineamidohydrolase. As certain names reveal, the disease occurs due to adeficiency of acid ceramidase (also known as N-acylsphingosineamidohydrolase, ASAH). The lack of the enzyme results in an accumulationof non-sulfonated acid mucopolysaccharide in the neurons and glialcells. Patients with the disease usually die before the age of 2 years.

Metachromatic leukodystrophy (MLD) is a genetic disorder caused by adeficiency of the enzyme arylsulfatase A. It is one of a group ofgenetic disorders called the leukodystrophies that affect growth of themyelin sheath. There are three forms of MLD: late infantile, juvenile,and adult. In the late infantile form, which is the most common, onsetof symptoms begins between ages 6 months and 2 years. The infant isusually normal at birth, but eventually loses previously gainedabilities. Symptoms include hypotonia (low muscle tone), speechabnormalities, loss of mental abilities, blindness, rigidity (i.e.,uncontrolled muscle tightness), convulsions, impaired swallowing,paralysis, and dementia. Symptoms of the juvenile form begin betweenages 4 and 14, and include impaired school performance, mentaldeterioration, ataxia, seizures, and dementia. In the adult form,symptoms, which begin after age 16, may include impaired concentration,depression, psychiatric disturbances, ataxia, tremor, and dementia.Seizures may occur in the adult form, but are less common than in theother forms. In all three forms mental deterioration is usually thefirst sign.

Peliszaeus-Merzbacher disease (also known as perinatal sudanophilicleukodystrophy) is an X-linked genetic disorder that causes anabnormality of a proteolipid protein. The abnormality results in aninfant's death typically before the age of one year. There are no knowntreatments or cures for the disease.

Refsum disease (also referred to as phytanic acid oxidase deficiency,heredopathia atactica polyneuritiformis or hereditary motor and sensoryneuropathy IV, HMSN IV) is caused by mutations in the gene, whichencodes phytanoyl-CoA hydroxylase (PAHX or PHYH). The major clinicalfeatures are retinitis pigmentosa, chronic polyneuropathy and cerebellarsigns. Phytanic acid, an unusual branched chain fatty acid(3,7,11,15-tetramethyl-hexadecanoic acid) accumulates in the tissues andbody fluids of patients with the disease and is unable to be metaboliseddue to the lack of PAHX. Plasmapheresis performed once or twice monthlyeffectively removes the acid from the body and permits liberalization ofdietary restrictions limiting phytanic acid intake.

Prion related conditions include Gerstmann-Straussler disease (GSD),Creutzfeldt-Jakob disease (CJD), familial fatal insomnia and aberrantisoforms of the prion protein can act as infectious agents in thesedisorders as well as in kuru and scrapie (a disease found in sheep). Theterm prion derives from “protein infectious agent” (Prusiner, Science216: 136-44, 1982). There is a proteolytic cleavage of the prion relatedprotein (PRP) which results in an amyloidogenic peptide that polymerisesinto insoluble fibrils.

Salla disease and other types of sialurias are diseases involvingproblems with sialic acid storage. They are autosomal recessiveneurodegenerative disorders that may present as a severe infantile form(i.e., ISSD) or as a slowly progressive adult form that is prevalent inFinland (i.e., Salla disease). The main symptoms are hypotonia,cerebellar ataxia and mental retardation. These conditions and diseasesare also contemplated for palliative or ameliorating treatments.

Other conditions that result in demyelination include post-infectiousencephalitis (also known as acute disseminated encephalomyelitis, ADEM),meningitis and injuries to the spinal cord. The compositions andconjugates disclosed herein are also contemplated for use in treatingthese other demyelinating conditions.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention. Unless otherwise stated, alltemperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning. ACN = acetonitrile bs = broad singlet d = Doublet dd =doublet of doublets Et₃N = triethylamine g = Grams h and hr = Hour HPLC= High performance (or pressure) liquid chromatography kg = kilogram kDa= kilodalton L = Liter m = multiplet M = Molar mg = milligram min =Minute mL = milliliter mm = millimeter mM = millimolar mmol = millimol s= Singlet sat. = saturated t = Triplet TFA = trifluoroacetic acid TLC ortlc = thin layer chromatography Ts = Tosyl μL = microliter μg =microgram μm = micron or micrometer

General Methods: Proton (¹H) and carbon (¹³C) nuclear magnetic resonancespectra (NMR) were obtained using a Gemini 2000 or Bruker Avance 300spectrometer. The presence of the polyethylene glycol (PEG) protons canbe detected by a large, broad singlet at 3.6 ppm. The integration ofthis signal can vary depending on the size of the PEG moiety. Presenceof the conjugated VLA-4 antagonist can also be detected in the ¹H NMRspectra of conjugates. Thin layer chromatography was performed onpre-coated sheets of silica 60 F₂₅₄ (EMD 15341-1) or pre-coated MKC18Fsilica 60 Å (Whatman 4803-110). Mass spectrometry was performed on anAgilent mass spectrometer (LC/MSD VL) in positive ion single quad mode.

HPLC Methods for PEG Products and PEG Conjugates:

Preparative reverse phase HPLC was performed using a Varian Prep Star(Model SD -1) module with a Varian UV detector set at 210 nm. Method A:Samples of PEG products and PEG conjugates were purified using reversephase HPLC on a Vydac C18, 300 Å pore size column (250 mm×21.2 mm),typically using a gradient of 35-50% ACN+0.1% TFA in 100 min at 20mL/min. Method B: Samples of PEG products and conjugates were purifiedusing reverse phase HPLC on a Vydac C18, 300 Å pore size column (250mm×50 mm), typically using a gradient of 35-50% ACN+0.1% TFA in 100 minat 60 mL/min.

Method C: The purity of PEG products and conjugates was confirmed viareverse phase analytical HPLC using an Agilent Series 1100 Quaternarysystem equipped with a Waters Symmetry 300 Å pore size, 3.51μ C18 column(150 mm×4.6 mm), using a gradient of 40-50% ACN w/0.1% TFA at a flowrate of 1.5 mL/min. and coupled to an Agilent 1100 variable wavelengthdetector set at 210 nm and a Sedex 75 evaporative light scatteringdetector (40° C., gain=5)

PEG Reagents: PEG starting materials were acquired through NOFCorporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku,Tokyo 150-6019) or Nektar Therapeutics (150 Industrial Road, San Carlos,Calif. 94070) as follows: 40 kDa 4-arm PEG alcohol (NOF Cat. SunbrightPTE40000); 40 kDa 3-arm PEG alcohol (NOF Cat. Sunbright GL400).

Example 1

Sodium hydroxide (10 g, 0.25 m) is dissolved in water (300 ml). To thissolution 4-nitrophenylalanine (50.3 g, 0.22 m) is added and stirreduntil complete dissolution. To the resulting solution the sodiumcarbonate (28.8 g, 0.26 m) is added and stirred suspension is cooled inan ice bath to +8° C. Benzyl chloroformate (44.7 g, 0.26 m) is addeddropwise with vigorous stirring, maintaining internal temperature in +60to +9° C. range. The mixture is stirred at +6° C. for additional 1 hr,transferred to the separatory funnel and washed with ether (2×150 ml).Aqueous phase is placed in a large Erlenmeyer flask (2L) and iscautiously acidified with dil. aq. HCl to pH=2 and extracted with ethylacetate (4×500 ml). The combined extracts are washed with water anddried with MgSO4. The solution is filtered and filtrate evaporated,residue is dissolved in ethyl acetate (150 ml) and diluted with hexane(500 ml). Crystalline material is filtered off and rinsed with coldsolvent, air dried to give Cbz-4-nitrophenylalanine, 75 g (99.5% yield).¹H-NMR, DMSO-d₆, (δ): 12.85 (bs, 1H), 8.12 (d, 2H, J=9 Hz), 7.52 (d, 2H,J=9 Hz), 7.30 (m, 5H), 4.95 (s, 2H), 4.28 (m, 1H), 3.32 (bs, 1H), 3.10(m, 2H).¹³C-NMR (δ): 173.1, 156.3, 146.6, 137.3, 130.8, 128.5, 128.0,127.8, 123.5, 65.6, 55.1, 36.6. MS (m/z): 367.1 [M+23].

The Cbz-4-nitrophenylalanine (75 g, 0.22 m) is dissolved in dioxane (300ml). The resulted stirred solution is cooled in Dry Ice bath to −20° C.(internal). The liquefied isobutylene (approx. 290 ml) is added followedby conc. sulfuric acid (35 ml) added in three equal portions, 30 minapart. The addition of acid is a very exothermic process, accompanied bysubstantial degree of polymerization. Efficient mechanical stirring isessential at this stage. Resulted mixture is stirred for 20 hr, allowingto warm up to ambient temperature then is cautiously poured into sat.aq. sodium carbonate solution (2L) and diluted with ethyl acetate (600ml). Organic layer is separated and aqueous layer is extracted withethyl acetate (2×200 ml). Combined extracts are washed with water anddried with sodium sulfate. The solution is filtered and evaporated todryness. The residue is taken up in ethyl acetate/hexane mixture (500ml; 1:1) and filtered through plug of silica gel (ca. 2×2 in). Thesilica is rinsed with an additional amount of the same solvent (2 Ltotal) and the filtrates are evaporated to give fully protected4-nitrophenylalanine as a viscous oil, 73 g (83% after two steps).¹H-NMR, CDCl₃, (δ): 8.12 (d, 2H, J=8.4 Hz), 7.36 (m, 7H), 5.35 (m, 1H),5.10 (m, 2H), 4.57 (m, 1H), 3.31 (m, 2H), 1.43 (s, 9H). ¹³C-NMR, CDCl₃,(δ): 169.7, 155.3, 146.9, 143.9, 136.0, 130.2, 128.4, 128.2, 128.0,123.3, 82.9, 66.9, 54.7, 38.2, 31.4, 27.8, 13.9. MS (m/z): 423.1 [M+23].

Protected 4-nitrophenylalanine (73 g, 0.18 m) is dissolved in ethanol(500 ml) and platinum oxide catalyst (1.5 g) is added. The resultingsolution is vigorously stirred in hydrogen atmosphere (50-60 psi) atambient temperature until further hydrogen adsorption ceased (3 hr). Thecatalyst is filtered off and the filtrate is evaporated to dryness, theresidue is taken up in ethyl acetate (200 ml) and filtered through plugof silica gel (2×2 in) using ethyl acetate-hexane mixture (3:2, 2L) torinse silica. The filtrate is concentrated to approx. 200 ml and hexane(500 ml) is added. The crystalline product is filtered off, rinsed withcold solvent and air-dried. Yield -56 g, 84%. ¹H-NMR, CDCl₃, (δ): 7.30(bs, 5H), 6.92 (d, 2H, J=8.1 Hz), 6.58 (d, 2H, J=8.1 Hz), 5.21 (m, 1H),5.10 (d, 2H, J=2.1 Hz), 4.46 (m, 1H), 3.59 (bs, 2H), 2.97 (s, 2H, J=5.4Hz), 1.42 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.6, 145.1, 136.3, 130.2,128.3, 127.9, 125.6, 115.0, 81.9, 66.6, 55.2, 37.4, 27.8 MS (m/z): 393.1[M+23].

Example 2

The product of Example 1, 4-aminophenylalanine, (20 g, 0.054 m) wasdissolved in ethanol (200 ml) and treated with Hunig's base (21 g, 0.162m, 3 eq) and 2-chloro-3-nitropyridine (10.3 g, 0.65 m, 1.2 eq). Resultedsolution was stirred under nitrogen atmosphere and heated to reflux for24 hr. LC analysis indicated presence of small amount of unreactedamine. The small additional amount of chloronitropyridine (1.1 g, 0.13eq) was added and reflux continued for another 24 hr. Reaction mixturewas cooled and evaporated to dryness. Residue was dissolved in ethylacetate (600 ml) and obtained solution was washed with water (1×200 ml),dil. aq. citric acid (0.2 N, 2×200 ml), brine (1×200 ml) and dried withsodium sulfate. Solids were filtered off and filtrate evaporated to give37 g of deep-red oil, containing expected product contaminated withexcess of chloronitropyridine. Impure product was purified by flashchromatography (Biotage 75L system) eluting with ethyl acetate:hexane(3:17) mixture. Fractions containing pure product were combined andevaporated to give deep-red, viscous oil, 26 g (99%). ¹H-NMR, CDCl₃,(δ): 10.10 (s, 1H), 8.49 (m, 2H), 7.57 (d, 2H, J=9 Hz), 7.35 (bs, 5H),7.19 (d, 2H, J=9 Hz), 6.84 (m, 1H), 5.30 (m, 1H), 5.13 (d, 2H, J=3 Hz),4.57 (m, 1H), 3.11 (m, 2H), 1.45 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.4,155.5, 155.1, 150.0, 136.7, 136.3, 135.4, 132.4, 129.9, 128.5, 128.3,128.0, 127.9, 122.2, 113.7, 82.2, 66.7, 55.1, 37.7, 27.8, 20.9. MS(m/z): 493.1 [M+1], 515.1 [M+23].

The red nitro compound (26 g, 0.054 m) was dissolved in THF (350 ml) andplatinum oxide catalyst (1.35 g) was added. Resulted mixture wasvigorously stirred under hydrogen atmosphere (50-60 psi) until hydrogenadsorption ceased (2 hr). Catalyst was filtered off and filtrateevaporated to dryness. Residue was dissolved in ethyl acetate (100 ml)and diluted with hexane (50 ml) till beginning of crystallization.Mixture was further diluted with ethyl acetate/hexane (1:1) mixture (300ml) and was left standing in refrigerator for 3 hr. Crystalline solidswere filtered off, rinsed with cold solvent and air-dried to giveproduct, 23 g, 94%. ¹H-NMR, CDCl₃, (δ): 7.81 (dd, 1H, J1=1.5 Hz, J2=4.8Hz), 7.33 (bs, 5H), 7.17 (d, 2H, J=8.4 Hz), 7.03 (d, 2H, J=8.4 Hz), 6.96(dd, 1H, J1=1.5 Hz, J2=7.5 Hz), 6.75 (dd, 1H, J1=5.0 Hz, J2=7.7 Hz),6.22 (s, 1H), 5.31 (m, 1H), 5.09 (bs, 2H), 4.50 (m, 1H), 3.41 (bs, 2H),3.02 (m, 2H), 1.43 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.6, 155.6, 145.5,140.21, 138.8, 136.3, 130.8, 129.9, 128.5, 128.3, 127.9, 123.4, 118.2,117.0, 82.0, 66.6, 55.2, 37.4, 27.9. MS (m/z): 407.1 [M−56], 463.1[M+1], 485.1 [M+23].

The aminopyridine (19 g, 0.041 m) was suspended in dichloromethane (200ml) and CDI (12 g, 0.074 m, 1.8 eq) was added. Resulted mixture wasstirred at ambient temperature for 20 hr. Reaction mixture was washedwith sat. aq. bicarbonate (2×100 ml), brine (1×100 ml) and dried withsodium sulfate. Solids were filtered off and filtrate evaporated todryness. Residue was dissolved in ethyl acetate (hot, 300 ml) and set tocrystallize. Crystalline product was filtered off, rinsed with coldethyl acetate and air-dried to give 19.9 g, 81% of the imidazolone.¹H-NMR, CDCl₃, (δ):10.63 (s, 1H), 8.06 (d, 1H, J=3 Hz), 7.66 (d, 2H, J=9Hz), 7.32 (m, 8H), 7.05 (m, 1H), 5.36 (m, 1H), 5.13 (s, 2H), 4.59 (m,1H), 3.17 (m, 2H), 1.45 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 170.4, 155.6,154.3, 143.8, 141.0, 136.2, 135.8, 131.8, 130.2, 128.3, 128.0, 125.9,122.2, 118.3, 116.0, 82.4, 66.8, 55.0, 37.7, 27.8. MS (m/z): 433.1[M−56], 489.2 [M+1], 511.2 [M+23].

Example 3

Pyridine-3-sulfonic acid (125 g, 0.78 m) was placed in a 1 L, 3-neckedflask equipped with mechanical stirrer, reflux condenser, thermometerand nitrogen inlet. Next, the phosphorus pentachloride (250 g, 1.19 m,1.5 eq) was added, followed immediately by the phosphorus oxychloride(330 ml, 3.8 m, 4.5 eq). The contents of flask were initially stirred atambient temperature for 30 min, then brought slowly to gentle reflux(internal temp. approx. 110° C.) over the next hour, kept at thistemperature for approx. 3.5 hr then allowed over the next 12 hr to coolback to ambient temperature. Gas evolution was observed during thistime. The volatiles were stripped under reduced pressure (at 12 mmHg/40°C.) and yellow semi-solid residue was diluted with DCM (1 L). The slurrywas poured slowly into the stirred, ice-cold sat. aq. bicarbonate,maintaining pH=7. Gas evolution was observed. The organic layer wasseparated and aqueous layer was back-extracted with DCM. The combinedextracts were washed with cold sat. aq. bicarbonate, brine and driedwith magnesium sulfate. The solids were filtered off and filtrateevaporated, leaving pyridine-3-sulfonyl chloride as a pale yellow, oilyliquid, 123 g (93% pure; 88% theory). ¹H-NMR, CDCl₃, (δ): 9.26 (d, 1H),8.98 (dd, 1H), 8.34 (m, 1H), 7.62 (m, 1H). ¹³C-NMR, CDCl₃, (δ): 155.3,147.4, 140.9, 134.6, 124.2.

MS (m/z): 178.0 [M+1].

L-penicillamine (150 g, 1.0 m) was dissolved with stirring in DI water(1500 ml), cooled in ice-bath to +8° C. and treated with formalin (150ml, 37% aq.). The reaction mixture was stirred at +8° C. for 2 hr, thencooling bath was removed and stirring continued for 12 hr. The clearsolution was concentrated under reduced pressure (14 mmHg/50°) leavingwhite residue. The solids were re-suspended, then dissolved in hot MeOH(2500 ml) and left standing at ambient temperature for 12 hr. The white,fluffy precipitate was filtered off and rinsed with cold methanol. Thefiltrate was concentrated and set to crystallize again. The collectedprecipitate was combined with the first crop and dried in vacuum ovenfor 24 hr at 55° C. at 45 mmHg. The yield of(R)-5,5-dimethylthiazolidine-4-carboxylic acid was 138 g (>99% pure; 86%theory). ¹H-NMR, DMSO-d₆, (δ): 4.25 (d, 1H), 4.05 (d, 1H), 3.33 (s, 1H),1.57 (s, 3H), 1.19 (s, 3H). ¹³C-NMR, DMSO-d₆, (δ):170.8, 74.4, 57.6,51.8, 28.9, 27.9. MS (m/z): 162.3 [M+1].

In a 4L reactor equipped with mechanical stirrer and thermometer, abuffer solution was prepared from potassium monobasic phosphate (43 g,0.31 m) and potassium dibasic phosphate (188.7 g, 1.08 m) in DI water(2L). The (R)-5,5-dimethylthiazolidine-4-carboxylic acid (107 g, 0.675m) was added and stirred until complete dissolution. The solution wascooled in an ice-bath to +8° C. A separately prepared solution ofpyridine-3-sulfonyl chloride (124 g, 0.695 m) in DCM (125 ml) was addeddropwise to the reactor with vigorous stirring over the 1 hr. The pH ofreaction mixture was monitored and after 4 hr, found to be pH=5 andadjusted to pH=6 by addition of solid bicarbonate. The mixture wasallowed to warm up to ambient temperature over 18 hr. The pH wasadjusted to 2 with dil. aq. sulfuric acid, stirred for 1 hr andprecipitated yellow solids were filtered off, rinsed with water toneutral. The solid cake was transferred into 2L Erlenmayer flask,suspended in DCM (500 ml) with occasional swirling for 5 min andfiltered off again. The filter cake was washed with DCM and air-dried.The yield of the title compound,(R)-5,5-dimethyl-3-(pyridin-3-ylsulfonyl)thiazolidine-4-carboxylic acidwas 148.9 g (98% pure; 73% theory). ¹H-NMR, DMSO-d₆, (δ): 9.05 (d, 1H),8.89 (m, 1H), 8.32 (m, 1H), 7.69 (m, 1H), 4.68 (q, 2H), 4.14 (s, 1H),1.35 (s, 3H), 1.29 (s, 3H). ¹³C-NMR, DMSO-d₆, (δ): 170.0, 154.3, 147.9,135.8, 134.1, 124.8, 72.6, 54.3, 50.2, 29.4, 25.0. MS (m/z): 303.2[M+1].

Example 4

The product of Example 2 (52 g, 0.106 m) was slurried in MeOH (450 ml),hydrogenation catalyst (8.7 g, 5% Pd/C, Degussa) was added and themixture was stirred under the hydrogen atmosphere (60 psi) until furtherabsorption ceased (ca. 2 hrs). THF (150 ml) was added to dissolveprecipitated solids and the solution was filtered through plug ofCelite, using DCM to rinse the filter. The filtrate was evaporated todryness, re-dissolved in DCM (300 ml) and stripped again. This operationwas repeated twice. The foamy solids were kept under high vacuum for 3hrs. The yield of title compound was 38.3 g (101% of theory). ¹H-NMR,CDCl₃, (δ): 8.08 (m, 1H), 7.56 (AB q, 4H), 7.37 (m, 1H), 7.06 (m, 1H),3.68 (m, 1H), 2.03 (m, 2H), 1.49 (s, 9H). ¹³C-NMR, CDCl₃, (δ): 173.8,154.6, 143.9, 141.0, 137.4, 131.5, 130.2, 126.1, 122.3, 118.0, 116.1,81.4, 56.0, 40.6, 27.9. MS (m/z): 299.3 [M−56], 355.4 [M+1], 377.4[M+23].

Example 5

The product of Example 4 (38.3 g, assume 0.106 m) was dissolved in DCM(500 ml) and treated successively with: N-methylmorpholine (27 g, 30 ml,0.266 m; 2.5 eq), HOBt (17.3 g, 0.128 m,; 1.2 eq), and the product ofExample 3 (33.8 g, 0.112 m; 1.06 eq). The resulting non-homogenoussolution was cooled in an ice-bath to +4° C. and treated with EDC (22.5g, 0.117 m; 1.1 eq) in one portion. The reaction mixture was stirred,allowing it to warm up to ambient temperature over the next 4 hr andthen for 18 hr more. The solvent was stripped and residue dissolved inethyl acetate (1.2L), washed with sat. aq. bicarbonate (2×250 ml), water(250 ml), brine (300 ml) and dried with magnesium sulfate. The solutionwas filtered and evaporated to dryness, leaving a light orange, viscousoil, 76 g (>>100%). The crude product was purified by flashchromatography on silica gel (Biotage 75L, in ethyl-acetate/methanol(3%) mixture. Fractions, containing pure product, were combined andevaporated to give 54 g of of the title compound (yield 83%). ¹H-NMR,CDCl₃, (δ): 10.37 (s, 1H), 9.11 (s, 1H), 8.87 (m, 1H), 8.19 (m, 1H),8.05 (m, 1H), 7.56 (AB q, 4H), 7.52 (m, 1H), 7.36 (m, 1H), 7.06 (m, 2H),4.83 (m, 1H), 4.58 (AB a, 2H), 3.96 (s, 1H), 3.19 (m, 2H), 1.49 (s, 9H),1.22 (s, 3H), 1.18 (s, 3H). ¹³C-NMR, CDCl₃, (δ): 169.7, 167.6, 153.9,148.4, 143.8, 140.9, 135.8, 135.6, 132.9, 131.9, 130.2, 125.9, 123.8,122.1, 118.0, 115.9, 82.8, 73.6, 60.3, 54.8, 53.7, 50.6, 37.8, 29.1,27.8, 23.9,14.1. MS (m/z): 583.3[M−56], 639.4 [M+1], 661.3 [M+23].

Example 6

To an ice chilled solution of ethyl trifluorobutyrate (15 g, 89 mmol)and ethyl formate (36 mL, 444 mmol) in THF (200 mL) under N₂ was added asolution of 1 M KOtBu in THF (107 mmol, 107 mL) over a 25-minute period.After 15 minutes the ice bath was removed and the reaction mixture wasstirred one hour at room temperature. Additional ethyl formate (18 mL,222 mmol) was then added and the reaction mixture was stirred overnight.The reaction mixture was concentrated and the residue partitionedbetween cold ether (100 mL) and cold water (300 mL). The pH of theaqueous phase was adjusted to 2 with concentrated HCl. The product wasextracted with dichloromethane (1×100 mL, 45×75 mL) and the combinedorganic extracts were washed with brine (1×100 mL), dried (MgSO₄),filtered, and concentrated to yield the title compound as thick oilwhich solidified upon standing, 10.2 g (58.5%). MS (m/z)=198 (M+H)⁺.

Example 7

To a solution of the product of Example 6 (10 g, 51 mmol) anddiethylguanidine sulfate (8.3 g, 25.2 mmol) in EtOH (60 mL) under N₂,was added NaOEt, 21% solution in EtOH (20.7 mL, 55.5 mmol) over a10-minute period. The reaction mixture was then heated at reflux for 5hours. The heterogeneous solution was cooled and poured into cold water(100 mL) to give a homogenous solution. The pH of the solution wasadjusted to approximately 3.5 with conc. HCl and 1 N HCl. A solidprecipitated from solution, which was collected by filtration. The lighttan solid was washed with water and air-dried, yielding 2.9 g, (23%) ofthe title compound. MS (m/z)=250 (M+H)⁺. ¹H NMR (300 MHz, CD₃OD) δ 7.65(br s, 1H), 3.55 (q, 4H), 3.30 (q, 2H), 1.25 (t, 6H).

Example 8

A flask was charged with the product of Example 7 (2.0 g, 8.02 mmol),DIEA (1.5 mL, 8.83 mmol), DMAP (0.98 g, 0.8 mmol), and dichloromethane(30 mL). The mixture was cooled to 0° C. and trifluoroacetic anhydride(1.5 mL, 8.83 mmol) was added. The reaction became homogeneous and wasstirred at 0° C. for 3 hours. The mixture was quenched with sat. NaHCO₃and extracted with dichlorormethane. The organic phase was washed with0.2 N citric acid, dried over Na₂SO₄, filtered, and concentrated invacuo to yield 2.87 g (94%) of the title compound as a brown solid. ¹HNMR (300 MHz, CDCl₃) δ 8.28 (s, 1H), 3.65-3.52 (m, 4H), 3.29-3.19 (q,2H), 1.22-1.17 (t, 6H).

Example 9

A solution of the product of Example 8 (1.3 g, 3.5 mmol),H-Phe(p-NO₂)OtBu (1.1 g, 4.2 mmol), and DIEA (0.9 mL, 5.3 mmol) in CH₃CN(14 mL) under N₂ was heated to reflux overnight. The next day additionalH-Phe(p-NO₂)OtBu (0.8 g, 3 mmol) was added and reflux was continued for3 days. The reaction mixture was then cooled and concentrated Theresidue taken-up in EtOAc (50 mL) and the organic portion washed with0.5 N KHSO₄ (3×50 mL), water (1×50 mL), brine (1×10 mL), dried (MgSO₄),filtered and concentrated to a brownish gum. The crude material waspurified by flash chromatography (5:1 hexanes/EtOAc) to yield 640 mg(38%) of the title compound as a golden gum. TLC: 3:1 hexanes/EtOAc,R_(f)=0.30, MS (m/z)=498 (M+H)⁺, ¹H NMR, (300 MHz, CDCl₃) δ 8.19 (d,2H), 7.80 (s, 1H), 7.25 (d, 2H), 5.19 (br d, 1H), 4.95 (q, 1H),3.70-3.50 (m, 4H), 3.45-3.25 (m, 2H), 3.10 (q, 2H), 1.40 (s, 9H), 1.05(t, 6H).

Example 10

The product of Example 9 (635 mg, 1.27 mmol) was dissolved in absoluteEtOH (5 mL) to which was added 35 mg of Pd/C, 10 wt %. The reaction wassubjected to hydrogenation (45 psi H₂) for 2.5 hours at which time 50mgs of Pd/C , 10 wt % was added and the reaction mixture again subjectedto hydrogenation (45 psi H₂) overnight. The reaction mixture wasfiltered through a pad of Celite and the filtrate was concentrated togive 452 mg (76%) of the title compound. MS (m/z)=468 (M+H)⁺, ¹H NMR(300 MHz, CDCl₃) δ 7.75 (s, 1H), 6.90 (d, 2H), 6.60 (d, 2H), 5.05 (br d,1H), 4.80 (q, 1H), 3.70-3.45 (m, 6H), 3.10-2.90 (m, 4H), 1.40 (s, 9H),1.15 (t, 6H).

Example 11

A solution of the product of Example 10 (598 mg, 1.28 mmol),2-chloro-3-nitropyridine (243 mg, 1.53 mmol), and DIEA (0.67 mL ,3.83mmol) in EtOH (5 mL) under N₂ was heated at reflux. The next day thereaction was cooled and additional 2-chloro-3-nitropyridine (40 mg, 0.25mmol) and DIEA (0.11 mL, 0.60 mmol) was added and the reaction washeated at reflux for one day. The reaction mixture was then concentratedand the residue taken-up in EtOAc (20 mL). The organic phase was washedwith water (2×20 mL). The combined aqueous washes was back extractedwith EtOAc (2×10 mL). The combined organic extracts were washed with 0.2N citric acid (3×20 mL), water (1×10 mL), sat. NaHCO3 (3×20 mL), brine(1×10 mL), dried (MgSO4), filtered and stripped to an orange gum. Thecrude product was purified by flash chromatography eluting with 4:1hexanes/EtOAc (R_(f)=0.14) to yield 610 mg (81%) of the title compoundas a red oil. MS (m/z)=590 (M+H)⁺, ¹H NMR (300 MHz, CDCl₃) δ 10.10 (s,1H), 8.55 (d, 1H), 8.50 (m, 1H), 7.79 (s, 1H), 7.75 (d, 2H), 7.15 (d,2H), 6.80 (q, 1H), 5.10 (br d, 1H), 4.90 (m, 1H), 3.70-3.45 (m, 4H),3.25 (m, 2H), 3.10 (q, 2H), 1.40 (s, 9H), 1.10 (t, 6H).

Example 12

To a solution of the product of Example 11 (610 mg, 1.03 mmol) inabsolute EtOH (5 mL) was added 60 mg of Pd/C, 10 wt %. The mixture wassubjected to hydrogenation (45 psi H₂) overnight. The next day thereaction mix was filtered through Celite and the filtrate concentratedto give 500 mg (87%) of the title compound. MS (m/z)=560 (M+H)⁺, ¹H NMR(300 MHz, CDCl₃) δ 7.85 (d, 2H), 7.80 (s, 1H), 7.20 (d, 2H), 7.05 (d,2H), 7.00 (d, 1H), 7.75 (m, 1H), 6.20 (br s 1H), 5.15 (br s, 1H), 4.85(m, 1H), 3.75-3.45 (m, 4H), 3.40 (br s, 2H), 3.15 (m, 2H), 3.05 (q, 2H),1.40 (s, 9H), 1.15 (t, 6H).

Example 13

A solution of the product of Example 12 (141 mg, 0.250 mmol) and CDI (62mg, 0.378 mmol) in CH₂Cl₂ (3 mL) was stirred overnight. The next dayadditional CDI (30 mg, 0.185 mmol) was added and the reaction wasstirred another day. The reaction mixture was then concentrated andtaken-up in EtOAc (10 mL) and the organic portion washed with 0.2 Ncitric acid (3×5 mL), water (1×5 mL), sat. NaHCO₃ (3×5 mL), brine (1×5mL), dried (MgSO₄), filtered and concentrated to yield 69 mg (47%) thetitle compound as a foam which was used without further purification. MS(m/z)=586 (M+H)⁺, ¹H NMR (300 MHz, CDCl₃) δ 8.20 (br s, 1H), 8.05 (d,1H), 7.80 (s, 1H), 7.65 (d, 2H), 7.90 (m, 3H), 7.05 (m, 1H), 5.15 (br d,1H), 4.95 (m, 1H), 3.70-3.45 (m, 4H), 3.25 (app d, 2H), 3.10 (q, 2H),1.40 (s, 9H), 1.15 (t, 6H).

Example 14

To a solution of 4,6-dichloro-5-aminopyrimidine (5.0 g , 30.7 mmol) inDMSO (30 mL) was added Na₂S.9H₂O (7.4 g, 30.8 mmol). The mixture wasstirred at room temperature overnight. Water (40 mL) was then added tothe mixture and the solution evaporated under reduced pressure toapproximately 6 mL. To this solution was added conc. HCl (0.5 mL) andwater to precipitate the product. The solution was filtered and theorange solid was washed with water and dried to afford 4.3 g (86%) ofthe title compound. ¹H NMR (300 MHz, DMSO-d₆) δ 5.84 (2H, s), 7.79 (1H,s), 14.37 (1H, br s); MS(m/z): MH⁺=162.

Example 15

To the product of Example 14 (4.3 g , 26 mmol) dissolved in conc. NH₄OH(4 mL) was added EtOH (40 mL). To this solution, Raney Nickel (excess)was added in portions. The reaction was stirred at room temperatureovernight and then heated at 80° C. for 2 hrs. The mixture was filteredthrough Celite and the filtrate concentrated. The crude product waspurified by flash chromatography on silica using EtOAc/hexanes to afford1.6 g (47%) of the title compound as a yellow solid. ¹H NMR (300 MHz,DMSO-d₆) δ 5.90 (2H, s), 8.20 (2H, s); MS(m/z) MH⁺=130.

Example 16

To the product of Example 15 (0.51 g, 3.9 mmol) in MeOH (20 mL) and HOAc(0.5 mL) was added CH₃CHO (0.52 mL, 9.2 mmol). Then NaBH₃CN (590 mg, 9.2mmol) was added in one portion. The reaction was stirred at roomtemperature overnight and additional HOAc, CH₃CHO, and NaBH₃CN wereadded. The reaction was stirred overnight, concentrated, and the residuewas taken up in EtOAc and sat. NaHCO₃. The separated aqueous layer wasback extracted with EtOAc. The combined organic layer was dried andconcentrated to a residue. The residue was dissolved in MeOH and treatedwith HOAc, CH₃CHO and NaBH₃CN as described above. Following the work upprocedure described above the crude product was purified by flashchromatography on silica using EtOAc/hexanes, to afford 0.35 g (57%) ofthe title compound as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 1.35 (3H,q, J=12 Hz), 3.29 (2H, m), 4.21 (1H, bs), 8.04 (1H, s), 8.36 (1H, s);MS(m/z): MH⁺=158.

Example 17

To the product of Example 16 (70 mg, 0.45 mmol) dissolved in DMF (1 mL)was added TEA (93 uL) and isonicotinoyl chloride (0.12 g, 0.67 mmol).The reaction mixture was stirred at room temperature for 2 days and thenpartitioned between EtOAc and sat. NaHCO₃. The separated aqueous layerwas back extracted with EtOAc. The combined organic layer was dried andconcentrated to give 67 mg (57%) of the title compound which was usedwithout further purification. ¹H NMR (300 MHz, CDCl₃) δ 1.26 (3H),3.65-3.69 (1H), 4.21 (1H), 7.17 (2H), 8.43 (1H), 8.54 (2H), 8.86 (1H)Note: ¹H NMR shows evidence of rotamers as demonstrated of broadness ofall peaks; MS(m/z): MH⁺=263.

Example 18

To a solution of the product of Example 17 (0.11 g, 0.42 mmol) and theproduct of Example 8 (0.135 g, 0.38 mmol) in IPA (2.5 ml) was added DIEA(0.35 ml, 1.9 mmol). The reaction mixture was stirred in a sealed tubeat 130° C. for 2 days. The crude mixture was concentrated and the oilwas purified by flash column chromatography with a solvent gradient of0-10% MeOH in CH₂Cl₂ to yield the title compound as an oil. ¹H NMR (300MHz, CDCl₃) δ 1.16 (1.2H, m), 1.26-1.31 (1.8H, m), 1.50-1.53 (9H, d, J=9Hz), 3.0 (1H, m), 3.2 (0.8H, m), 3.36 (1.2H, m), 4.12-4.18 (1.2H, m),4.96-5.10 (0.8H, m), 5.80-5.95 (1H, m), 6.93-6.96 (1H, m), 7.07 (1H, m),7.31-7.45 (5H, m), 7.66-7.75 (3H, m), 8.06 (1H, m), 8.44-8.51 (2H, m);HPLC/MS: single peak at 1.29 min, MH⁺=581.

Example 19

To 2,4-dichloro-5-nitropyrimidine (2.0 g, 10.3 mmol) in MeOH (7 mL) at0° C. under N₂ was added NaOMe (0.5 M in MeOH, 25 mL) dropwise. Afterthe addition was completed, the reaction mixture was stirred at 0 C for15 min. Then diethylamine (5 mL) was added and the mixture was stirredat rt overnight. The reaction mixture was concentrated and the residuewas partitioned between EtOAc and H₂O. The organic layer was dried andconcentrated to a residue which was purified by flash chromatography onsilica using EtOAc/Hexanes, to afford the title compouns as an off whitesolid (1.1 g, 4.9 mmol, 47% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.26 (6H,t, J=6.6 Hz), 3.70 (4H, m), 4.08 (3H, s), 9.01 (1H, s); HPLC/MS:MH⁺=227.

Example 20

The product of Example 19 (1.19 g, 4.9 mmol) in MeOH/EtOAc (1:1, 20 mL)was reduced with Pd/C (5% degussa, 0.5 g) and H₂ (50 psi) in a Parrshaker overnight. The reaction mixture was filtered and the filtratedwas concentrated under reduced pressure to afford the title compound asa solid (0.85 g, 4.3 mmol, 88.5% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.18(6H, t, J=6.9 Hz), 3.03 (2H, br), 3.57 (6H, t, J=6.9 Hz), 3.96 (3H, s),7.71 (1H, s); HPLC/MS: MH⁺=197.

Example 21

To the product of Example 20 (0.85 g, 4.3 mmol) in CH₂Cl₂ (15 mL) andTEA (1.4 mL, 10 mmol) was added isonicotinyl chloride HCl salt (1.13 g,6.3 mmol). After 15 min, TLC showed no starting material. The mixturewas extracted between EtOAc and sat. NaHCO₃. The aqueous layer waswashed with EtOAc twice. The combined organic layers were washed withsat. NaHCO₃ and brine. It was dried over MgSO₄ and filtered. Thefiltrate was concentrated to give the title compound as a brown solid(1.3 g, 4.3 mmol, 100% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.20 (6H, t,J=6.9 Hz), 3.60 (4H, q, J=6.9 Hz), 3.96 (3H, s), 7.72 (2H, d, J=6.0 Hz),7.75 (1H, bs), 8.80 (2H, d, J=6.0 Hz), 8.89 (1H, s); HPLC/MS: MH⁺=302.

Example 22

To the product of Example 21 (100 mg, 0.33 mmol) in THF (1 mL) was addedKOtBu (1 M in THF, 0.5 mL) slowly followed by EtI (40 □L, 0.5 mmol). Thereaction mixture was stirred at rt overnight. TLC showed thedisappearance of the starting material. The mixture was partitionedbetween EtOAc and H₂O. The aqueous layer was washed with EtOAc. Thecombined organic layers were washed with sat. NaHCO₃ and brine. It wasdried and concentrated to give the title compound (90 mg, 0.27 mmol,83%) that was used without further purification. ¹H NMR (300 MHz, CDCl₃)δ 1.10 (9H, m), 3.47 (5H, m), 3.92 (1H, m), 7.14 (2H, d, J=6.0 Hz), 7.78(1H, bs), 8.44 (2H, d, J=6.0 Hz); HPLC/MS: MH⁺=330.

Example 23

To the product of Example 22 (200 mg, 0.61 mmol) in DMF (4 mL) was addedEtSNa (66 mg, 0.79 mmol) and the reaction mixture was heated at 100 Cfor 1 hr. LC/MS showed starting material still present. Another portionof NaSEt (66 mg, 0.79 mmol) was added and the reaction heated foranother 2 hr. LC/MS showed product only. DMF was removed under reducedpressure and H₂O (10 mL) was added followed by conc. HCl (0.132 mL).Evaporating of the solvent left a residue. It was dissolved in EtOH andfiltered. The filtrate was concentrated to to yield the title compound(190 mg, 100%) that was used without further purification. ¹H NMR (300MHz, CD₃OD) δ 1.24 (9H, m), 3.60 (4H, m), 3.60-4.00 (2H, br), 8.12 (3H,d, J=5.7 Hz), 8.92 (2H, d, J=5.7 Hz); HPLC/MS: MH⁺=316.

Example 24

To the product of Example 23 (70 mg, 0.22 mmol) in POCl₃ (3 mL) at rtwas added diethylaniline (30 μL). The reaction mixture was heated to 100C for 30 min. Then it was concentrated. The residue was partitionedbetween EtOAc and H₂O. The organic layer was washed with H₂O twice. Thenit was dried and concentrated to give the title compound (50 mg, 0.15mmol, 68%) and used for the next reaction without further purification.HPLC/MS: MH⁺=334

Example 25

To a solution of the product of Example 24 (50 mg, 0.15 mmol) and theproduct of Example 8 (60 mg, 0.17 mmol) in IPA (0.75 mL) was added DIEA(0.15 mL, 0.8 mmol). The reaction mixture was stirred in a sealed tubeat 130 degrees for 7 days. The crude mixture was concentrated and theresidue was purified by preparative HPLC and silica gel flashchromatography to yield an off white solid (10 mg). ¹H NMR (300 MHz,CDCl₃) 61.10-1.30 (9H, m), 1.48 (4.5H, s), 1.51 (4.5H, s), 2.80-3.38(3H, m), 3.53 (4H, m), 4.05-4.30 (1H, m), 4.83 (0.5H, m), 4.96 (0.5H,m), 5.15-5.50 (1H, m), 6.95-7.10 (2H, m), 7.25-7.50 (5H, m), 7.69 (0.5H,d, J=8.4 Hz), 7.76 (0.5H, d, J=8.4 Hz), 8.08 (1H, d, J=5.1 Hz), 8.51(2H, m), 8.83 (0.5H, br), 8.95 (0.5H, br);

HPLC/MS: MH+=652.

Example 26

Procedure A

Example 26A

The 40 kDa 3-arm PEG alcohol (0.25 g, 0.00625 mmol), the product fromExample 5 (0.04 g, 0.056 mmol), and triphenylphosphine (0.025 g, 0.094mmol) were dried by azeotropic distillation from toluene (5 mL). Half ofthe volume was distilled over (2.5 mL), and the mixture was cooled toroom temperature. CH₂Cl₂ (0.5 mL) was added to make the reactionhomogeneous. Diethylazodicarboxylate (0.015 mL, 0.094 mmol) was addeddrop-wise and the reaction stirred for 48 hours. HPLC Method C showedthe complete disappearance of the starting PEG alcohol. The reaction wasconcentrated in vacuo to yield the t-butyl ester Example 26A as a whitesolid.

Example 26B

Example 26A (0.2 g, 0.005 mmol) was dissolved in formic acid (3 mL) andheated at 40° C. for 24 hours. The reaction was concentrated in vacuoand was purified according to HPLC Method A to yield 0.1 g (48%) ofExample 26B as a white solid. HPLC Method C determined the conjugate tobe >99% pure (retention time=8.1 minutes). ¹H NMR (CDCl₃) δ 9.08 (bs,3H), 8.84 (bs, 3H), 8.18-8.16 (d, 3H), 8.02-8.00 (d, 3H), 7.67-7.61 (m,6H), 7.47-7.38 (m, 9H), 7.08-7.04 (m, 3H), 6.91 (m, 3H), 4.88 (m, 3H),4.62-4.49 (dd, 6H), 4.13 (m, 6H), 3.64 (bs, 5919H PEG), 3.23 (m, 6H),1.25-1.24 (d, 18H).

Procedure B

A mixture of GL400 Sunbright PEG (50.0 g, NOF lot# M4N594), the productfrom Example 5 (7.19 g, 9 eq vs. GL400, 3 eq/hydroxyl), andtriphenylphosphine (2.95 g, 9 eq) was taken up in toluene (300 mL) anddistilled to azeotropically remove water. The mixture was cooled to roomtemperature and an additional amount of the remaining toluene wasremoved via rotary evaporation. The mixture was re-dissolved in drydichloromethane (180 mL) and cooled in an ice bath.Diisopropylazodicarboxylate (DIAD, 2.27 g, 2.17 mL, 9 eq) was added viasyringe drive over 1 hour. The mixture was stirred in the ice batch 1.5h whereupon HPLC analysis indicated complete conversion to Example 26A.

The viscous reaction mixture was slowly added via a narrow-necked funnelwith stirring to a mixture of 75:25 MTBE/IPA (4.0 L) and allowed to stir1 hour. The precipitate was collected via vacuum filtration, washed withMTBE (200 mL), and dried under vacuum to give purified Example 26A (51.5g.).

Example 26A (51.4 g) was taken up into formic acid (250 mL) and heatedat near reflux for 1.0 h whereupon HPLC analysis indicated thedeprotection was complete. The mixture was cooled to room temp and aportion of the formic acid (−100 mL) was removed by rotary evaporation.The mixture was diluted with methylene chloride (50 mL). The viscousreaction mixture was then added with stirring to a mixture of 75:25MTBE/IPA (4.0 L) and allowed to stir 45 min (Note 4). The precipitatewas collected via vacuum filtration, washed with MTBE (300 ml) and driedunder vacuum (<1 Torr, 24 h) to yield Example 26B (50.0 g).

This material was taken up in methylene chloride (400 mL) and filteredthrough a sintered-glass Buchner funnel (“polish filtration”). Thefiltrate was concentrated via rotary evaporation to a volume of ˜200 mLand slowly added with stirring to a 75:25 mixture of MTBE/IPA (4.0L).The precipitate was collected via vacuum filtration, washed with MTBE(300 ml) and dried under vacuum (<1 Torr, 3d) to yield Example 26B (48.8g, -94%).

Similar methods were used to synthesize the following conjugates:

Example 27

Example 27

40 kDa 4-arm PEG alcohol was coupled to the product of Example 5 anddeprotected to final product using similar methods as with Example 26.The product was purified according to HPLC Method A. HPLC Method Cdetermined the conjugate to be >95% pure (retention time=7.5-8.1minutes). ¹H NMR (CDCl₃) δ9.08 (bs, 4H), 8.84 (bs, 4H), 8.18-8.16 (d,4H), 8.02-8.00 (d, 4H), 7.67-7.61 (m, 8H), 7.47-7.38 (m, 12H), 7.08-7.04(m, 4H), 6.91 (m, 4H), 4.88 (m, 4H), 4.62-4.49 (dd, 8H), 4.13 (m, 8H),3.64 (bs, 10101H PEG), 3.23 (m, 8H), 1.25-1.24 (d, 24H).

Example 28

Example 28

40 kDa 3-arm PEG alcohol was coupled to the t-butyl ester product fromExample 18 (shown below) and deprotected to final product using methodssimilar to those of Example 26. The product was purified according toHPLC Method A. HPLC Method C determined the conjugate to be >95% pure(retention time=7.3 minutes). ¹H NMR (CDCl₃) δ 8.66 (bs, 3H), 8.44 (bs,3H), 8.04-8.02 (d, 3H), 7.75-7.30 (m, 24H), 7.10-7.06 (m, 3H), 6.93 (s,3H), 5.60-5.50 (m, 3H), 4.15 (m, 6H), 3.66 (bs, 4270H PEG), 3.00 (m,3H), 3.40-3.20 (m, 6H), 1.27 (d, 9H).

Example 29

Example 29 Example 29A

The 40 kDa 3-arm PEG alcohol (0.00625 mmol), the product from Example 13(0.056 mmol), and triphenylphosphine (0.094 mmol) are dried byazeotropic distillation from toluene (5 mL). Half of the volume isdistilled over (2.5 mL), and the mixture is cooled to room temperature.CH₂Cl₂ (0.5 mL) is added to make the reaction homogeneous.Diethylazodicarboxylate (0.094 mmol) is added drop-wise and the reactionstirred for 48 hours. The reaction is concentrated in vacuo to yield thet-butyl ester Example 29A.

Example 29B

Example 29A (0.005 mmol) is dissolved in formic acid (3 mL) and heatedat 40° C. for 24 hours. The reaction is concentrated in vacuo and ispurified according to HPLC Method A to yield Example 29B.

Example 30

Example 30

40 kDa 3-arm PEG alcohol is coupled to the t-butyl ester product fromExample 25 and deprotected to final product using similar methods aswith Example 26. The product is purified according to HPLC Method A.

REFERENCES

The following publications, patents and patent applications are cited inthis application as superscript numbers:

-   1 Hemler and Takada, European Patent Application Publication No.    330,506, published Aug. 30, 1989-   2 Elices, et al., Cell, 60:577-584 (1990)-   3 Springer, Nature, 346:425-434 (1990)-   4 Osborn, Cell, 62:3-6 (1990)-   5 Vedder, et al., Surgery, 106:509 (1989)-   6 Pretolani, et al., J. Exp. Med., 180:795 (1994)-   7 Abraham, et al., J. Clin. Invest., 93:776 (1994)-   8 Mulligan, et al., J. Immunology, 150:2407 (1993)-   9 Cybulsky, et al., Science, 251:788 (1991)-   10 Li, et al., Arterioscler. Thromb., 13:197 (1993)-   11 Sasseville, et al., Am. J. Path., 144:27 (1994)-   12 Yang, et al., Proc. Nat. Acad. Science (USA), 90:10494 (1993)-   13 Burkly, et al., Diabetes, 43:529 (1994)-   14 Baron, et al., J. Clin. Invest., 93:1700 (1994)-   15 Hamann, et al., J. Immunology, 152:3238 (1994)-   16 Yednock, et al., Nature, 356:63 (1992)-   17 Baron, et al., J. Exp. Med., 177:57 (1993)-   18 van Dinther-Janssen, et al., J. Immunology, 147:4207 (1991)-   19 van Dinther-Janssen, et al., Annals. Rheumatic Dis., 52:672    (1993)-   20 Elices, et al., J. Clin. Invest., 93:405 (1994)-   21 Postigo, et al., J. Clin. Invest., 89:1445 (1991)-   22 Paul, et al., Transpl. Proceed., 25:813 (1993)-   23 Okarhara, et al., Can. Res., 54:3233 (1994)-   24 Paavonen, et al., Int. J. Can., 58:298 (1994)-   25 Schadendorf, et al., J. Path., 170:429 (1993)-   26 Bao, et al., Diff., 52:239 (1993)-   27 Lauri, et al., British J. Cancer, 68:862 (1993)-   28 Kawaguchi, et al., Japanese J. Cancer Res., 83:1304 (1992)-   29 Kogan, et al., U.S. Pat. No. 5,510,332, issued Apr. 23, 1996-   30 International Patent Appl. Publication No. WO 96/01644-   31 Thorselt, et al., U.S. Pat. No. 6,489,300, issued Dec. 3, 2002    and Konradi, et al., U.S. Pat. No. 66,492,372, issued Dec. 10, 2002.

All of the above publications, patents and patent applications areherein incorporated by reference in their entirety to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A process for the preparation of conjugates of active compoundscomprising: a) reacting a polymeric alcohol with a nucleophilic activecompound having an acidic hydrogen under Mitsunobu conditions; and b)isolating the conjugate.
 2. A process according to claim 1, where theconditions comprise a trivalentphosphine and an azodicarbonyl compound.3. A process for the preparing a conjugate of the formula I:

B is a bio-compatible polymer moiety optionally covalently attached to abranched-arm hub molecule; q is from about 1 to about 100; A at eachoccurrence is independently a compound of formula II

or a pharmaceutically acceptable salt thereof, wherein J is selectedfrom: a) a group of formula (a):

wherein R³¹ is a covalent bond to the polymer moiety which optionallycomprises a linker, or R³¹ is —H, R^(31′), —NH₂, —NHR^(31′) or—N(R^(31′))₂, —NC₃-C₆cyclic, —OR^(31′), —SR^(31′), wherein each R^(31′)is independently an optionally substituted straight or branchedC₁-C₆alkyl, optionally substituted C₃-C₆cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, and R³² is acovalent bond to the polymer moiety which optionally comprises a linker,or R³² is —H, —NO₂, haloalkyl or the group —N(MR⁴¹)R⁴² wherein M is acovalent bond, —C(O)— or —SO₂—, R⁴¹ is R^(41′), N(R^(41′))₂, or—OR^(41′), wherein each R^(41′) is independently hydrogen, an optionallysubstituted straight or branched C₁-C₆alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedheterocyclic or an optionally substituted heteroaryl, wherein optionalsubstitutions are halide, C₁-C₆alkyl, or —OC₁-C₆alkyl, and R⁴² ishydrogen or R^(41′); and b) a group of formula (b):

wherein R is selected from the group consisting of a covalent bond tothe polymer moiety, amino, hydroxyl, substituted amino, alkyl, alkyloxy,aryloxy, heteroaryloxy, heterocyclyloxy, thiol, arylthio,heteroarylthio, heterocyclylthio and substituted alkyl wherein eachamino, substituted amino, alkyl and substituted alkyl is optionallycovalently bound to the polymer moiety wherein, in each case, thepolymer moiety optionally comprises a linker which covalently links thepolymer moiety; Ar¹ is selected from the group consisting of aryl,substituted aryl, heteroaryl and substituted heteroaryl wherein each ofaryl, substituted aryl, heteroaryl and substituted heteroaryl isoptionally covalently bound to the polymer moiety wherein the polymermoiety optionally comprises a linker which covalently links the polymermoiety to Ar¹; Ar² is selected from the group consisting of aryl,substituted aryl, heteroaryl and substituted heteroaryl wherein each ofaryl, substituted aryl, heteroaryl and substituted heteroaryl isoptionally covalently bound to the polymer moiety wherein the polymermoiety optionally comprises a linker which covalently links the polymermoiety to Ar²; X is selected from the group consisting of —NR¹—, —O—,—S—, —SO—, —SO₂ and optionally substituted —CH₂— where R¹ is selectedfrom the group consisting of hydrogen and alkyl; T is selected from: a)a group of formula (c)

wherein Y is selected from the group consisting of —O— and —NR¹— whereinR¹ is selected from the group consisting of hydrogen and alkyl; W isselected from the group consisting of a covalent bond to a polymermoiety which optionally comprises a linker and —NR²R³ wherein R² and R³are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, and where R² and R³, together with the nitrogen atombound thereto, form a heterocyclic ring or a substituted heterocyclicring wherein each of alkyl, substituted alkyl, heterocyclic andsubstituted heterocyclic is optionally covalently bound to a polymermoiety which further optionally comprises a linker; m is an integerequal to 0, 1 or 2; n is an integer equal to 0, 1 or 2; and b) a groupof formula (d)

wherein G is an optionally substituted aryl or optionally substitutedheteroaryl 5 or 6 membered ring containing 0 to 3 nitrogens, whereinsaid aryl or heteroary optionally further comprises a covalent bond to apolymer moiety which optionally comprises a linker; R⁶ is a covalentbond to a polymer moiety which optionally comprises a linker, or R⁶ is—H, alkyl, substituted alkyl, or —CH₂C(O)R⁷, wherein R¹⁷ is —OH, —OR¹⁸,or —NHR⁸, wherein R¹⁸ is alkyl, substituted alkyl, aryl or substitutedaryl; R⁵⁵ is selected from the group consisting of amino, substitutedamino, alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy,aryloxy and substituted aryloxy, and —OH; provided that: A. at least oneof R, Ar¹, Ar², and T contains a covalent bond to the polymer moiety; B.when R is covalently bound to the polymer moiety, n is one and X is not—O—, —S—, —SO—, or —SO₂—; C. when X is —O— or —NR¹—, then m is two; andD. the conjugate of formula I has a molecular weight of no more than100,000; comprising the steps of: c) adding a polymeric alcohol offormula Ia

wherein B is as described above, to a nucleophile of formula H-Nu in thepresence of a trivalentphosphine of the formula PR₃ and a dialkylazodicarboxylate to form the compound of formula I, wherein Nu is aradical of formula A described above; and isolating the compound offormula I.
 4. The process according to claim 1, wherein only one of J,Ar², and T contains a covalent bond to a polymer moiety.
 5. The processaccording to claim 1, wherein, q is an integer of from 1 to about
 20. 6.The process according to claim 1, wherein q is an integer of from 1 toabout
 8. 7. The process according to claim 1, wherein A at eachoccurrence is independently a compound of formula IIa


8. The process according to claim 1, wherein A at each occurrence isindependently a compound of formula IIb:


9. The process according to claim 1, wherein A at each occurrence isindependently a compound of IIc:


10. The process of claim 1, wherein the nucleophilic active compound isa compound of the formula

wherein J is selected from: a) a group of formula (a):

wherein R³¹ is a covalent bond to the polymer moiety which optionallycomprises a linker, or R³¹ is —H, R^(31′), —NH₂, —NHR^(31′) or—N(R^(31′))₂, —NC₃-C₆cyclic, —OR^(31′), —SR^(31′), wherein each R^(31′)is independently an optionally substituted straight or branchedC₁-C₆alkyl, optionally substituted C₃-C₆cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, and R³² is acovalent bond to the polymer moiety which optionally comprises a linker,or R³² is —H, —NO₂, haloalkyl or the group —N(MR⁴¹)R⁴² wherein M is acovalent bond, —C(O)— or —SO₂—, R⁴¹ is R^(4′), N(R^(4′))₂, or —OR^(4′),wherein each R^(41′) is independently hydrogen, an optionallysubstituted straight or branched C₁-C₆alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedheterocyclic or an optionally substituted heteroaryl, wherein optionalsubstitutions are halide, C₁-C₆alkyl, or —OC₁-C₆alkyl, and R⁴² ishydrogen or R^(41′); and b) a group of formula (b):

wherein R is selected from the group consisting of a covalent bond tothe polymer moiety, amino, hydroxyl, substituted amino, alkyl, alkyloxy,aryloxy, heteroaryloxy, heterocyclyloxy, thiol, arylthio,heteroarylthio, heterocyclylthio and substituted alkyl wherein eachamino, substituted amino, alkyl and substituted alkyl is optionallycovalently bound to the polymer moiety wherein, in each case, thepolymer moiety optionally comprises a linker which covalently links thepolymer moiety; Ar¹ is selected from the group consisting of aryl,substituted aryl, heteroaryl and substituted heteroaryl wherein each ofaryl, substituted aryl, heteroaryl and substituted heteroaryl isoptionally covalently bound to the polymer moiety wherein the polymermoiety optionally comprises a linker which covalently links the polymermoiety to Ar¹; Ar² is selected from the group consisting of aryl,substituted aryl, heteroaryl and substituted heteroaryl wherein each ofaryl, substituted aryl, heteroaryl and substituted heteroaryl isoptionally covalently bound to the polymer moiety wherein the polymermoiety optionally comprises a linker which covalently links the polymermoiety to Ar²; X is selected from the group consisting of —NR¹—, —O—,—S—, —SO—, —SO₂ and optionally substituted —CH₂— where R¹ is selectedfrom the group consisting of hydrogen and alkyl; T is a group carryingan acidic hydrogen; and R⁵⁵ is a acid protecting group.
 11. The of claim10, the nucleophilic active compound has the formula:

where R⁵⁵ is an acid protecting group.
 12. A process according to claim11, where R⁵⁵ is C₁-C₆ alkoxy.
 13. The process according to claim 11wherein m is 1, X is S, and R at each occurrrence is independentlyselected from hydroxyl, alkyloxy, alkyl, or a covalent bond to thepolymer moiety.
 14. The process according to claim 12 wherein n is 2,and R at both occurrences is methyl.
 15. The process of claim 1, whereinthe nucleophilic active compound has the formula:

where R⁵⁵ is an acid protecting group.
 16. A process according to claim15, where R⁵⁵ is C₁-C₆ alkoxy.
 17. The process according to claim 15wherein G is pyridinyl, R31 is hydrogen or dialkylamino, and R32 issulfonamide, amide, or urea.
 18. A process according to claim 1, whereinthe nucleophilic active compound is:


19. A process according to claim 1, wherein the polymer alcohol is


20. A process according to claim 1, wherein the polymer alcohol isselected from the group in column A and the nucleophilic active compoundis selected from the group in column B: A B

(total Mw of conjugate is about 41,500)

(total Mw of conjugate is about 42,000)

(total Mw of conjugate is about 41,500)


21. The process according to claim 1 wherein the polymeric alcohol isadded to the nucleophile of formula H-Nu in the presence of thetrivalentphosphine and the dialkyl azodicarboxylate in a at least onesolvent.
 22. The process according to claim 21 wherein the dialkylazodicarboxylate is selected from the group consisting ofdiethyldiazocarboxylate, diisopropylazodicarboxylate,4-methyl-1,2,4-triazolidine-3,5-dione,N,N,N′,N′-tetramethylazodicarboxamide, azodicarboxylic aciddipiperidide, bis(N-4-methylpiperazin-1-yl)azodicarboxamide,dimorpholinoazodicarboxamide and di-tert-butyl azodicarboxylate.
 23. Theprocess according to claim 22 wherein the dialkyl azodicarboxylate isselected from the group consisting of diethyldiazocarboxylate,diisopropylazodicarboxylate, 4-methyl-1,2,4-triazolidine-3,5-dione,N,N,N′,N′-tetramethylazodicarboxamide, azodicarboxylic acid dipiperidideand di-tert-butyl azodicarboxylate.
 24. The process according to claim21 wherein the solvent is a chlorinated solvent or an ether solvent. 25.The process according to claim 24 wherein the solvent is adichloromethane or tetrahydrofuran.
 26. The process according to claim21 wherein the trivalentphosphine is selected from the group consistingof triphenylphosphine, trimethylphosphine, triethylphosphine,tributylphosphine 1,2-bis-(diphenylphosphino)ethane, a polymer boundphosphine and a water soluble phosphine.
 27. The process according toclaim 26 wherein the trivalent phosphine is triphenylphosphine.
 28. Aprocess according to claim 1, wherein the conjugate is prepared usingpolymer alcohol/nucleophilic active compound combination 1, 2, or 3:Combi- nation Polymer Alcohol Nucleophilic Active Compound 1

(total Mw of conjugate is about 41,500) 2

(total Mw of conjugate is about 42,000) 3

(total Mw of conjugate is about 41,500)